U.S. patent application number 17/252577 was filed with the patent office on 2021-09-09 for methods of detection of mechanically-activated platelet activation and uses thereof.
The applicant listed for this patent is Arizona Board of Regents on Behalf of the University of Arizona, Naing BIJAJ, The Research Foundation for the State University of New York. Invention is credited to Naing Bijaj, Danny Bluestein, Michael Dicaro, Louise Hecker, Yana Roka-Moiia, Marvin J. Slepian.
Application Number | 20210277357 17/252577 |
Document ID | / |
Family ID | 1000005655549 |
Filed Date | 2021-09-09 |
United States Patent
Application |
20210277357 |
Kind Code |
A1 |
Slepian; Marvin J. ; et
al. |
September 9, 2021 |
METHODS OF DETECTION OF MECHANICALLY-ACTIVATED PLATELET ACTIVATION
AND USES THEREOF
Abstract
Biochemical markers that can be measured to determine the level
of mechanical activation of a population of platelets are provided,
and their use to prepare molecular signatures thereof are provided.
The markers include phosphatidylserine, thrombin, integrin
GPIIb/IIIa activation, glycoprotein GP Ib, P-selectin; platelet
size; microparticle generation, or lipidomic profile of the
membrane. The disclosed markers, measurement thereof, and
signatures composed therefrom can be used in a variety of methods
of patient selection, treatment monitoring, treatment selection,
including both devices and active agents, and methods of treating
subjects in need thereof, particularly humans. Examples of such
methods include, but are not limited to, reducing shear-activated
platelets; selecting a blood-contacting medical device; selecting
between two or more medical devices; identifying a subject at a
risk of developing a thrombogenic event; monitoring the
prophylactic treatment of a subject; selecting a mechanoceutical
agent; and delivering an agent to a subject.
Inventors: |
Slepian; Marvin J.; (Tucson,
AZ) ; Roka-Moiia; Yana; (Tucson, AZ) ;
Bluestein; Danny; (Stony Brook, NY) ; Bijaj;
Naing; (Tucson, AZ) ; Dicaro; Michael;
(Tucson, AZ) ; Hecker; Louise; (Tucson,
AZ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BIJAJ; Naing
Arizona Board of Regents on Behalf of the University of Arizona
The Research Foundation for the State University of New
York |
Tucson
Tucson
Albany |
AZ
AZ
NY |
US
US
US |
|
|
Family ID: |
1000005655549 |
Appl. No.: |
17/252577 |
Filed: |
June 17, 2019 |
PCT Filed: |
June 17, 2019 |
PCT NO: |
PCT/US2019/037528 |
371 Date: |
December 15, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62685703 |
Jun 15, 2018 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 13/00 20130101;
A61K 35/19 20130101; C12N 5/0644 20130101; G01N 33/68 20130101 |
International
Class: |
C12N 5/078 20060101
C12N005/078; A61K 35/19 20060101 A61K035/19; C12N 13/00 20060101
C12N013/00; G01N 33/68 20060101 G01N033/68 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with government support under Grant
Nos. U01 HL131052 and U01 EB012487 awarded by NIH. The government
has certain rights in the invention.
Claims
1. A method of creating a molecular signature of platelets
activated by one or more mechanical stimuli comprising measuring a
composite of at least two of (a) phosphatidylserine, (b) thrombin,
(c) integrin GPIIb/IIIa activation, (d) glycoprotein GP Ib, (e)
P-selectin; (f) platelet size; (g) microparticle generation, and
(h) lipidomic profile of the membrane, optionally the composite
comprises at least three, at least four, at least five, at least
six, at least seven, or eight of the measurements of (a)-(h).
2. The method of claim 1, wherein the one or more mechanical
stimuli are shear, vibration, audible sound, ultrasound, acoustic
stimulation, or pressure, or a combination thereof.
3. The method of claim 1, wherein the platelets are isolated from a
blood sample of a subject.
4. A molecular signature of mechanically activated platelets
comprising two or more molecular features selected from the group
consisting of: (a) an elevated phosphatidylserine level on the
external surface of the platelets relative to a level of a control;
(b) an elevated thrombin level on the external surface of the
platelets relative to the level of the control; (c) an integrin
GPIIb/IIIa activation level on the external surface of the
platelets, wherein the level is not increased compared to the level
of the control; (d) a glycoprotein GP Ib level on the external
surface of the platelets, wherein the level is not increased
compared to the level of the control; (e) a P-selectin level on the
external surface of the platelets, wherein the level is not
increased compared to the level of the control; (f) a decrease in
platelet size relative to the platelet size of the control; (g) an
increase in microparticle generation relative to the microparticle
generation of the control, and (h) a change in membrane lipidomic
profile compared to the membrane lipidomic profile of the
control.
5. A method of reducing shear-activated platelets in the blood of a
subject comprising (a) selecting a subject comprising platelets
with the molecular signature of claim 4, and (b) administering to
the subject a pharmaceutical composition comprising one or more
mechanoceuticals in an amount effective to reduce the level of
phosphatidylserine, the level of thrombin, and/or the level of
microparticle generation; to increase in platelet size; or to
change membrane lipidomic profile; or a combination thereof;
optionally without affecting the level of integrin GPIIb/IIIa
activation, the level of integrin GP Ib, and/or the level of
P-selectin in the platelets of the subject.
6. An ex vivo method for selecting a blood-contacting medical
device for implanting into a subject in need thereof comprising:
(a) modeling a shear stress profile of a blood-contacting medical
device; (b) reproducing the shear stress profile of blood flow in a
microfluidic device; (c) flowing a sample collected from the
subject through the microfluidic device; wherein the sample
comprises platelets optionally separated from a blood sample
obtained from the subject; (d) creating a molecular signature of
the platelets comprising measuring a composite of at least two of
(a) phosphatidylserine, (b) thrombin, (c) integrin GPIIb/IIIa
activation, (d) glycoprotein GP Ib, (e) P-selectin; (f) platelet
size; (g) microparticle generation, and (h) lipidomic profile of
the membrane, optionally wherein the composite comprises at least
three, at least four, at least five, at least six, at least seven,
or eight of the measurements of (a)-(h); and (e) selecting the
blood-contacting medical device if the level of phosphatidylserine,
on the external surface of platelets is lower than (a) of the
molecular signature of claim 4, if the level of thrombin on the
external surface of platelets is lower than (b) of the molecular
signature of claim 4, if the level of microparticle generation is
lower than (g) of the molecular signature of claim 4; if the
platelet size is greater than (f) of the molecular signature of
claim 4; or a combination thereof; optionally the level of integrin
GPIIb/IIIa activation, the level of integrin GP Ib, and/or the
level of P-selectin in the platelets of the subject is not
different than each of (c), (d), and (e), respectively, of the
molecular signature of claim 4.
7. The method of claim 6, wherein step (a) comprises one or more
of: using a device emulating thrombogenicity, producing a
probability density function of the blood-contacting medical
device, and determining trajectories for individual particles
flowing through the blood-contacting medical device.
8. The method of claim 6, wherein the molecular signature of the
platelets of step (d) is similar to a molecular signature of a
control.
9. A method of selecting between two or more medical devices,
comprising carrying out the method of claim 6 on each of the two or
more devices individually, and selecting the device with the lowest
level of phosphatidylserine on the external surface of the
platelets, the lowest level of thrombin on the external surface of
the platelets, and/or the lowest level of microparticle
generation.
10. A method of treating a subject in need thereof comprising
implanting into the subject a medical device selected according to
the method of claim 6.
11. The method of claim 6, wherein the medical device is a
mechanical circulatory support device.
12. A method for identifying a subject at a risk of developing a
thrombogenic event comprising (a) creating a molecular signature of
the platelets in the blood sample collected from a subject
comprising measuring a composite of at least two of (a)
phosphatidylserine, (b) thrombin, (c) integrin GPIIb/IIIa, (d)
glycoprotein GP Ib, (e) P-selectin; (f) platelet size; (g)
microparticle generation, and (h) lipidomic profile of the
membrane, optionally wherein the composite comprises at least
three, at least four, at least five, at least six, at least seven,
or eight of the measurements of (a)-(h); and (b) identifying the
subject as positive for developing a thrombogenic event if the
molecular signature of step (a) is similar to the molecular
signature of claim 4.
13. The method of claim 11, further comprising (c) administering to
subjects identified as positive for developing a thrombogenic event
one or more mechanoceuticals in an effective amount to reduce the
level of phosphatidylserine, the level of thrombin, and/or the
level of microparticle generation; to increase in platelet size; to
change membrane lipodomic profile; or a combination thereof;
optionally without affecting the level of integrin GPIIb/IIIa, the
level of integrin GP Ib, and/or the level of P-selectin in the
platelets of the subject.
14. A method of monitoring the prophylactic treatment of a subject
comprising: (a) creating a molecular signature of the platelets in
the blood sample collected before treatment from a subject
comprising measuring a composite of at least two of (a)
phosphatidylserine, (b) thrombin, (c) integrin GPIIb/IIIa
activation, (d) glycoprotein GP Ib, (e) P-selectin; (f) platelet
size; (g) microparticle generation, and (h) lipidomic profile of
the membrane, optionally wherein the composite comprises at least
three, at least four, at least five, at least six, at least seven,
or eight of the measurements of (a)-(h), (b) treating the subject
with a mechanoceutical one or more times, (c) creating a molecular
signature of the platelets in the blood sample collected after step
(b) from the subject comprising measuring a composite of at least
two of (a) phosphatidylserine, (b) thrombin, (c) integrin
GPIIb/IIIa activation, (d) glycoprotein GP Ib, (e) P-selectin; (f)
platelet size; (g) microparticle generation, and (h) lipidomic
profile of the membrane, optionally wherein the composite comprises
at least three, at least four, at least five, at least six, at
least seven, or eight of the measurements of (a)-(h), (d) repeating
step (b) if the level of phosphatidylserine, the level of thrombin,
the level of microparticle generation is reduced; the platelet size
is increased; and/or the membrane lipidomic profile is changed;
optionally the level of integrin GPIIb/IIIa activation, the level
of integrin GP Ib, and/or level of P-selectin in the platelets of
the subject is not affected.
15. The method of claim 14, wherein if the level of
phosphatidylserine, the level of thrombin, the level of
microparticle generation is not reduced; the platelet size is not
increased; and/or the membrane lipidomic profile is not changed;
(e) increasing the dose, the frequency of administration of the
mechanoceutical, or a combination thereof, or administering to the
subject a different mechanoceutial.
16. The method of claim 15, further comprising repeating steps (c)
and (d).
17. A method for selecting a mechanoceutical agent comprising: (a)
reproducing a shear stress profile of blood flow in a microfluidic
device; (b) flowing a sample collected from the subject through the
microfluidic device in the presence and absence of a test agent,
wherein the sample comprises platelets separated from a blood
sample obtained from a subject; (c) creating a molecular signature
of the platelets in the presence and absence of the test agent
comprising measuring a composite of at least two of (a)
phosphatidylserine, (b) thrombin, (c) integrin GPIIb/IIIa
activation, (d) glycoprotein GP Ib, (e) P-selectin; (f) platelet
size; (g) microparticle generation, and (h) lipidomic profile of
the membrane, optionally wherein the composite comprises at least
three, at least four, at least five, at least six, at least seven,
or eight of the measurements of (a)-(h); and (d) selecting the test
agent as a mechanoceutical if the level of phosphatidylserine on
the external surface of platelets, the level of thrombin on the
external surface of platelets, and/or the level of microparticle
generation is lower in the presence of the test agent than in the
absence of the test agent.
18. A method for delivering an agent to a subject comprising (a)
administering to the subject one or more mechanoceuticals one or
more times, and (b) creating a molecular signature of the platelets
in the blood sample collected after step (a) from the subject
comprising measuring a composite of at least two of (a)
phosphatidylserine, (b) thrombin, (c) integrin GPIIb/IIIa
activation, (d) glycoprotein GP Ib, (e) P-selectin; (f) platelet
size; (g) microparticle generation, and (h) lipidomic profile of
the membrane, optionally wherein the composite comprises at least
three, at least four, at least five, at least six, at least seven,
or eight of the measurements of (a)-(h).
19. The method of claim 18, further comprising subsequently
repeating step (b) and (c) repeating step (a) if the level of
phosphatidylserine, the level of thrombin, and/or the level of
microparticle generation is reduced; the platelet size is
increased; and/or the membrane lipidomic profile is changed;
optionally the level of integrin GPIIb/IIIa activation and/or level
of P-selectin in the platelets of the subject is not affected.
20. The method of claim 18, wherein if the level of
phosphatidylserine, the level of thrombin, and/or the level of
microparticle generation is not reduced; the platelet size is not
increased; and/or the membrane lipodomic profile is not changed;
(d) increasing the dose, the frequency of administration of the
mechanoceutical, or a combination thereof, or administering to the
subject a different mechanoceutial.
21. The method of claim 18, wherein the subject has a
blood-contacting medical device implanted in the body prior to step
(a).
22. The method of claim 18, further comprising (e) implanting a
blood-contacting medical device into the subject, wherein steps (e)
and (a) are simultaneous or wherein step (a) is subsequent to step
(e).
22. The method of claim 1, wherein the control is a population of
resting platelets.
23. The method of claim 1, wherein the subject is a human.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of and priority to U.S.
Ser. No. 62/685,703 filed Jun. 15, 2018, which is hereby
incorporated by reference in its entirety.
FIELD OF INVENTION
[0003] The present invention relates to the field of
cardiology.
BACKGROUND OF THE INVENTION
[0004] Mechanical circulatory support (MCS) in form of ventricular
assist devices (VADs) and total artificial hearts is utilized as
bridging-to decision, recovery and destination life-support therapy
of advanced heart failure. MCS while clinically effective remains
limited by thrombotic complications, such as ischemic stroke and
pump thrombosis, driven by mechanical platelet activation under
extremely high shear stress (up to 10000 s.sup.-1) existing within
VAD-operated blood flow. Pump thrombosis, being complex to diagnose
and requiring aggressive clot prevention therapy, carries
significant morbidity and leads to multiple readmission and
reoperations. According to INTERMACS annual report, pump
replacement markedly reduced life-expectancy for 1-year survivals
of 65% after the second implant and only 50% after the third
implant (Starling et al., N. Engl. J. Med., vol. 370, no. 1, pp.
33-40, January (2014)).
[0005] Beyond blood contact with foreign surfaces, MCS
device-related thrombosis is mainly potentiated by
supraphysiological flow phenomena that promotes thrombus formation
via direct mechanical activation of circulating platelets in free
flow. A variety of platelet responses are reported to be driven by
high shear stress, e.g. von Willebrand factor (vWF) binding and
integrin .alpha.IIb.beta.3 activation, signal transduction and
platelet shape change, and releasing of secretory granules and
platelet aggregation. According to traditional paradigm of arterial
thrombogenesis as thrombus formation in high shear environment,
shear-triggered vWF binding to glycoprotein (GP) complex Ib-IX-V is
needed for initialization of rapid platelet adhesion under elevated
blood flow, subsequent .alpha.IIb.beta.3 activation and
stabilization of platelet aggregates. GPIb-IX-V complex is
considered to play a principal role in transmembrane transduction
of mechanical activation signal inside the cell. Representing some
similarities with classic biochemical activation, SMPA indeed
results in other dramatic events more likely to be pro-apoptotic.
Pathological shear levels (over 100 dyn/cm.sup.2) were demonstrated
to cause platelet caspase activation, mitochondrial potential
dissipation, plasmatic membrane depolarization, cell shrinkage and
fragmentation (Brown et al., Trans. Am. Soc. Artif. Intern. Organs,
vol. 21, pp. 35-9, (1975)). Over the last few years understanding
of SMPA phenomena have been expanded beyond the boundaries of a
platelet mechanical damage theory. Even so, the molecular
mechanisms of mechanoreception and intracellular
mechanotransduction of the signal triggering SMPA, as well as the
holistic picture of events underlying SMPA by itself are not fully
understood.
[0006] To evaluate platelet function alterations in patients with
MCS, a standard panel of platelet activation markers are monitored,
among them membrane integrins (.alpha.IIb.beta.3, GP Ib, CD31)
exposed on platelet surface and secretory granule proteins
(platelet factor 4, .beta.-thromboglobulin, membrane bound and
soluble forms of P-selectin & CD40) released after their
activation.
[0007] Currently, observations from the clinical experience are
controversial and no specific molecular marker of SMPA correlating
with platelet function tests and thrombotic outcomes has been
reported. For instance, early studies of bridging patients with
either pulsatile (Berlin Heart and Novacor system) or axial flow
(MicroMed DeBakey) LVADs have indicated that increased platelet
factor 4 and .beta.-thromboglobulin levels, which are similar to
coagulation parameters within late post-operative period,
corresponded to thromboembolic events reported later on
(Himmelreich et al., ASAIO J., vol. 41, no. 3, pp. M790-4, Koster
et al., Ann. Thorac. Surg., vol. 70, no. 2, pp. 533-7, August
(2000)). After external LVAD (Thoratec Corp.) implantation, the
platelet surface P-selectin expression remained increased,
indicating persistent platelet activation, but GP Ib and GP IIbIIIa
activation levels were unaffected (Mi Houel et al., "Platelet
activation and aggregation profile in prolonged external
ventricular support)).
[0008] Independently of length of support, anti-platelet and
anticoagulant regiments, axial flow VADs Jarvik 2000* and
HeartMateII implanted for destination therapy did not cause
detectable increase in soluble platelet activation markers
(P-selectin and CD40 ligand) even considering high rotational speed
of the devices (Loffler et al., J. Thorac. Cardiovasc. Surg., vol.
137, no. 3, pp. 736-741, March (2009), Slaughter et al., Int. J.
Artif. Organs, vol. 34, no. 6, pp. 461-468, June (2011)). In
contrast, other studies examined platelet functions during
HeartMate II and HeartWare long-term support, which did not find
differences in CD41, CD42 and CD62 levels; whereas platelet
aggregation and VASP phosphorylation level, as platelet reactivity
index, were reportedly significantly increased (Birschmann et al.,
J. Hear. Lung Transplant., vol. 33, pp. 80-87, (2014)). Summarizing
clinical observation, the assessment of known platelet activation
markers during MCS failed to reflect platelet function alterations
caused by VAD-modified shear conditions. Despite of anticoagulant
and antithrombotic interventions, those functional tests still
indicate platelet activation and coagulation intensification which
might prerequisite/contribute (to) device-related thrombotic events
occurred afterwards.
[0009] To date, conventional assays of platelet activation have
limited ability to detect early platelet function alterations due
to mechanical activation and to distinguish such alterations from
those associated with biochemical platelet activation by soluble
agonists and adhesive proteins. So far, no specific molecular
marker of SMPA correlating with platelet function tests and
post-implantation thrombotic outcomes has been reported (A. Koster
et al., Ann. Thorac. Surg., 70(2):533-7 (2000), R. Mi Houel et al.,
J Thorac Cardiovasc Surg. 128(2):197-202 (2004). R. Radovancevic et
al., ASAIO J. 55(5):459-464 (2009), C. Loffler et al., J. Thorac.
Cardiovasc. Surg., 137(3):736-741 (2009), M. S. Slaughter, et al.,
Int. J. Artif. Organs, 34(6):461-468 (2011), I. Birschmann et al.,
J. Hear. Lung Transplant, 33:80-87 (2014)). Currently, non-specific
targeting of multiple signaling pathways of platelet biochemical
activation by traditional anti-platelet therapeutics within
antithrombotic regiments provides no benefits or even causes
side-effect associated comorbidity in VAD-supported patients.
[0010] There is a need for methods for prediction of device-related
thrombosis. There is a need for methods to accurately monitor of
antithrombotic therapy for VAD-supported patients.
[0011] Therefore, it is an object of the invention to provide
methods for monitoring platelet activation pathways, particularly
those associated with mechanically-mediated platelet
activation.
[0012] It is an object of the invention to provide platelet
activation pathways and biomarkers that can be used for diagnostic
and therapeutic purposes.
[0013] It is a further object of the invention to provide improved
methods for monitoring the disease activities and responses to
treatment for subjects with VADs.
SUMMARY OF THE INVENTION
[0014] Biochemical markers that can be measured to determine the
level of mechanical activation of a population of platelets are
provided. The markers include (a) phosphatidylserine, (b) thrombin,
(c) integrin GPIIb/IIIa, (d) glycoprotein GP Ib, (e) P-selectin;
(f) platelet size; (g) microparticle generation, and/or (h)
lipidomic profile of the membrane.
[0015] Methods of measuring the biochemical markers, and using the
measurements to prepare composite molecular signatures of the
platelets are also provided. The composites typically include
measurements of at least two, at least three, at least four, at
least five, at least six, at least seven, or eight of markers
(a)-(h).
[0016] A molecular signature of mechanically activated platelets
typically includes two or more molecular features selected from (a)
an elevated phosphatidylserine level on the external surface of the
platelets relative to a level of a control; (b) an elevated
thrombin level on the external surface of the platelets relative to
the level of the control; (c) an integrin GPIIb/IIIa level on the
external surface of the platelets, wherein the level is not
increased compared to the level of the control; (d) a glycoprotein
GP Ib level on the external surface of the platelets, wherein the
level is not increased compared to the level of the control; (e) a
P-selectin level on the external surface of the platelets, wherein
the level is not increased compared to the level of the control;
(f) a decrease in platelet size relative to the platelet size of
the control; (g) an increase in microparticle generation relative
to the microparticle generation of the control; and (h) a change in
membrane lipidomic profile compared to the membrane lipidomic
profile of the control.
[0017] The disclosed markers, measurement thereof, and signatures
composed therefrom can be used in a variety of methods of patient
selection, treatment monitoring, and treatment selection, including
both devices and active agents, and methods of treating subjects in
need thereof, particularly humans. Examples of such methods are
discussed in more detail below and include: reducing
shear-activated platelets in the blood of a subject; selecting a
blood-contacting medical device for implanting into a subject in
need thereof; selecting between two or more medical devices;
identifying and optionally treating a subject at a risk of
developing a thrombogenic event; monitoring the prophylactic
treatment of a subject; selecting a mechanoceutical agent; and
delivering an agent to a subject in need thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIGS. 1A-1F are line plots showing representative kinetic
curves of human platelet aggregation in gel-filtered platelets
(GFP) and platelet-rich plasma (PRP) induced by 10 uM ADP (FIG.
1A), 10 .mu.g/mL epinephrine (FIG. 1B), 32 .mu.M TRAP-6 (FIG. 1C),
1 U/mL thrombin (FIG. 1D), 100 .mu.g/mL collagen (FIG. 1E) and 1 mM
arachidonic acid (FIG. 1F).
[0019] FIGS. 2A-2H are graphs showing human platelet P-selectin
exposure induced by biochemical activation: FIGS. 2A-2G are
representative flow cytometry histogram plots showing distribution
of platelet P-selectin exposure detected by anti-CD62P-APC staining
in intact platelets (20.times.10.sup.3 cells/.mu.L) (FIG. 2A), in
gel-filtered platelets (GFP) incubated with ADP (FIG. 2B),
epinephrine (FIG. 2C), collagen (FIG. 2D), TRAP-6 (FIG. 2E),
thrombin (FIG. 2F), and arachidonic acid (FIG. 2G), along with 2.5
mM CaCl.sub.2 undisturbed for 10 min at room temperature.
CD62P-positive platelets were marked as P6 population. The number
of P6 populations of 4 independent experiments with different
donors are summarized, mean value+/-margins of error as error bars
are plotted in the bar graph (FIG. 2H). P values were calculated vs
intact platelets by one-way ANOVA: *--p.ltoreq.0.05,
**--p.ltoreq.0.01; for bars without asterisks, p>0.05.
[0020] FIGS. 3A-3F are graphs showing human platelet P-selectin
exposure induced by constant uniform shear: FIGS. 3A-3E are
representative flow cytometry histogram plots showing distribution
of platelet P-selectin exposure detected by anti-CD62P-APC staining
in intact platelets (FIG. 3A), in GFP (20.times.10.sup.3
cells/.mu.L) sheared utilizing the hemodynamic shearing device
(HSD) constant modes 30 dynes/cm.sup.2 (FIG. 3B), 50 dynes/cm.sup.2
(FIG. 3C), or 70 dynes/cm.sup.2 (FIG. 3D) for 10 mM at room
temperature, and in the positive control treated with sonication
(FIG. 3E). CD62P-positive platelets were marked as P6 population.
The number of P6 populations of 6 independent experiments with
different donors are summarized, mean value+/-margins of error as
error bars are plotted in the bar graph (FIG. 3F). P values were
calculated vs intact platelets by one-way ANOVA: *--p.ltoreq.0.05,
**--p.ltoreq.0.01; for bars without asterisks, p>0.05.
[0021] FIGS. 4A-4H are graphs showing platelet integrin
GPIIb/IIIa-activation induced by biochemical activation: FIGS.
4A-4G are representative flow cytometry histogram plots showing
distribution of activated GPIIb/IIIa on gel-filtered platelets
(GFP) detected using dual-staining with anti-CD41/CD61-FITC (PAC-1
clone, designed against the epitope that appears after GPIIb/IIIa
activation) & anti-CD41-APC (against .alpha.IIb, exposed in
non-activated and activated integrin) in intact platelets (FIG.
4A), in GFP (20.times.10.sup.3 cells/.mu.L) incubated with ADP
(FIG. 4B), epinephrine (FIG. 4C), collagen (FIG. 4D), TRAP-6 (FIG.
4E), thrombin (FIG. 4F), and arachidonic acid (FIG. 4G), along with
2.5 mM CaCl.sub.2 undisturbed for 10 min at room temperature.
Percentage of CD41/CD61-positive platelets out of total
CD41-positive platelets is marked as P3 population. The number of
P6 populations of 6 independent experiments with different donors
are summarized, mean value+/-margins of error as error bars are
plotted in the bar graph (FIG. 4H). P values were calculated vs
intact platelets by one-way ANOVA: *--p.ltoreq.0.05,
**--p.ltoreq.0.01; for bars without asterisks, p>0.05.
[0022] FIGS. 5A-5F are graphs showing human platelet integrin
GPIIb/IIIa-activation induced by constant uniform shear: FIGS.
5A-5E are representative flow cytometry histogram plots showing
destribution of GPIIb/IIIa of gel-filtered platelets (GFP) detected
using dual-staining with anti-CD41/CD61-FITC (PAC-1 clone, designed
against the epitope that appears after GPIIb/IIIa activation) &
anti-CD41-APC (against .alpha.IIb, exposed in non-activated and
activated integrin) in intact platelets (FIG. 5A), in GFP
(20.times.10.sup.3 cells/.mu.L) sheared utilizing HSD constant
modes 30 dynes/cm.sup.2 (FIG. 5B), 50 dynes/cm.sup.2 (FIG. 5C), or
70 dynes/cm.sup.2 (FIG. 5D) for 10 min at room temperature, and in
the positive control treated with sonication (FIG. 5E). Percentage
of CD41/CD61-positive platelets out of total CD41-positive
platelets is marked as P3 population. P6 populations of 6
independent experiments with different donors are summarized, mean
value+/-margins of error as error bars are plotted in the bar graph
(FIG. 5F). P values were calculated vs intact platelets by one-way
ANOVA: *--p.ltoreq.0.05, **--p.ltoreq.0.01; for bars without
asterisks, p>0.05.
[0023] FIGS. 6A-6C are bar graphs showing the number of platelets
expressing integrin GPIIb/IIIa (FIG. 6A), platelet fluorescence
intensity (FIG. 6B), and size distribution (FIG. 6C) of GFP
(20.times.10.sup.3 cells/.mu.L) sheared utilizing HSD constant
modes 30, 50, or 70 dynes/cm.sup.2 for 10 min at room temperature,
and in the positive control treated with sonication. Integrin
GPIIb/IIIa expression was detected using anti-CD41-APC. The bar
graphs represent 6 independent experiments with different donors,
mean value.+-.margins of error as error bars are plotted. P values
were calculated vs intact platelets by one-way ANOVA:
*--p.ltoreq.0.05, **--p.ltoreq.0.01; for bars without asterisks,
p>0.05.
[0024] FIGS. 7A-7H are graphs showing human platelet
phosphatidylserine (PS) externalization induced by biochemical
activation: FIGS. 7A-7G are representative flow cytometry histogram
plots showing distribution of platelet PS externalization detected
using FITC-bound annexin V in intact platelets (FIG. 7A), in GFP
(20.times.10.sup.3 cells/.mu.L) incubated with ADP (FIG. 7B),
epinephrine (FIG. 7C), collagen (FIG. 7D), TRAP-6 (FIG. 7E),
thrombin (FIG. 7F), and arachidonic acid (FIG. 7G), along with 2.5
mM CaCl.sub.2) undisturbed for 10 mM at room temperature. Annexin
V-positive platelets was marked as P3 population. The number of P3
populations of 6 independent experiments with different donors are
summarized, mean value+/-margins of error as error bars are plotted
in the bar graph (FIG. 7H). P values were calculated vs intact
platelets by one-way ANOVA: *--p.ltoreq.0.05, **--p.ltoreq.0.01;
for bars without asterisks, p>0.05.
[0025] FIGS. 8A-8G are graphs showing human platelet
phosphatidylserine externalization induced by constant uniform
shear: FIGS. 8A-8E are representative flow cytometry histogram
plots showing distribution of platelet PS externalization detected
using FITC-bound annexin V in intact platelets (FIG. 8A), in GFP
(20.times.10.sup.3 cells/.mu.L) sheared utilizing HSD constant
modes 30 dynes/cm.sup.2 (FIG. 8B), 50 dynes/cm.sup.2 (FIG. 8C), or
70 dynes/cm.sup.2 (FIG. 8D) for 10 mM at room temperature, and in
the positive control treated with sonication (FIG. 8E). Annexin
V-positive platelets were marked as P3 population. The number of P3
populations of 8 independent experiments with different donors are
summarized, mean value+/-margins of error as error bars are plotted
in the bar graph (FIG. 8F). P values were calculated vs intact
platelets by one-way ANOVA: *--p.ltoreq.0.05, **--p.ltoreq.0.01;
for bars without asterisks, p>0.05. FIG. 8G is a bar graph
showing the mean fluorescent intensity (MFI) of intact platelets,
shear-treated, and sonicated platelets ("70 dynes/cm2" vs. "Intact
platelets", ANOVA p<0.01).
[0026] FIGS. 9A-9B are bar graphs showing thrombin generation on
the surface of human platelets as measured with
prothrombinase-based platelet activation state (PAS) assay in
intact platelets, or GFP (20.times.10.sup.3 cells/.mu.L) stimulated
with biochemical activators including ADP, epinephrine, collagen,
TRAP-6, and arachidonic acid along with 2.5 mM CaCl.sub.2)
undisturbed for 10 mM at room temperature (FIG. 9A); or in GFP
(20.times.10.sup.3 cells/.mu.L) sheared utilizing HSD constant
modes 30, 50, or 70 dynes/cm.sup.2 for 10 mM at room temperature,
and in the positive control treated with sonication (FIG. 9B). The
bar graphs represent 4 independent experiments with different
donors, mean value.+-.margins of error as error bars are plotted. P
values were calculated vs intact platelets by one-way ANOVA:
*--p.ltoreq.0.05, **--p.ltoreq.0.01; for bars without asterisks,
p>0.05.
[0027] FIGS. 10A-10C are bar graphs showing phosphatidylserine
externalization (FIG. 10A), procoagulant activity (FIG. 10B),
P-selectin exposure (FIG. 10C), and of platelets recirculated
through the VAD-employed loop system. GFP (20.times.10.sup.3
cells/.mu.L) were recirculated through Heart Assist V-utilizing
model circulatory system for 1 hour at room temperature, timing GFP
samples were collected and processed immediately. On bar graphs
data of 4 (6 for C) independent experiments with different donors
are summarized, mean value.+-.margins of error as error bars are
plotted. P values were calculated vs intact platelets by one-way
ANOVA: *--p<0.05, **--p<0.01; for bars without asterisks,
p>0.05.
DETAILED DESCRIPTION OF THE INVENTION
[0028] Medical diagnosis involves consideration of a composite of
biomarkers. It has been found that significant differences in
certain biomarkers are associated with mechanically-activated
platelets compared to biochemical platelet activation by known
biochemical agonists. While a diagnosis of the cause of a
thrombogenic response in a subject can be made using only two of
the biomarkers described herein, the sensitivity of the diagnosis
increases the greater the number of biomarkers that are
present.
[0029] Platelets can be mechanically activated by a variety of
mechanical actions including shear, vibration, audible sound,
ultrasound, acoustic stimulation, pressure, or long-term storage in
blood bank environment.
[0030] Biomarkers for mechanically-activated platelets include one
or more of the following: an elevated phosphatidylserine level on
the external surface of the platelets relative to a level of a
control; an elevated thrombin level on the external surface of the
platelets relative to the level of the control; an integrin
GPIIb/IIIa level on the external surface of the platelets and
GPIIb/IIIa activation level, wherein these levels are not increased
compared to the level of the control; a glycoprotein GP Ib level on
the external surface of the platelets, wherein the level is not
increased compared to the level of the control; a P-selectin level
on the external surface of the platelets, wherein the level is not
increased compared to the level of the control.
[0031] Additional indicators of the presence of a polulation of
mechanically-activated platelets include one or more of the
following: significant (or excessive) microparticle generation,
change of lipidomic profile of the membrane (increase and decrease
of specific lipid species), early change in average platelet shape,
and decrease in average platelet size relative to a control.
I. Definitions
[0032] The term "microfluidic device" refers to a device comprising
fluidic structures and internal channels having microfluidic
dimensions. These fluidic structures may include chambers, valves,
vents, vias, pumps, inlets, nipples, and detection means, for
example. Generally, microfluidic channels are fluid passages having
at least one internal cross-sectional dimension that is less than
approximately 500 .mu.m to 1000 .mu.m and typically between
approximately 0.1 .mu.m and approximately 500 .mu.m. The
microfluidic flow regime is characterized by "Poiseuille" or
"laminar" flow (see, e.g., Staben et al. (2005) "Particle transport
in Poiseuille flow in narrow channels" Intl J Multiphase Flow
31:529-47, and references cited therein).
[0033] The term "mechanoceuticals" are generally molecules that
alter one or more physical properties of a cell (e.g., a platelet),
including but not limited to membrane fluidity/stiffness/stability,
membrane function, intracellular microfilament function,
intracellular microtubule function, intracellular fluid content and
tonicity, and submembrane assemblies.
II. Molecular Signatures of Mechanically Activated Platelets
[0034] Molecular signatures of mechanically activated platelets are
described. Platelets can be activated by chemical or mechanical
stimuli. Exemplary mechanical stimuli are shear, vibration, audible
sound (ultrasound), acoustic stimulation, pressure, and a
combination thereof.
[0035] Shear-mediated platelet activation (SMPA) is central in
thrombosis of implantable cardiovascular therapeutic devices.
Molecular signatures of SMPA are also provided. The terms "resting
platelets" and "intact platelets" are used interchangeable herein
and generally refer to unactivated platelets.
[0036] A. Molecular Markers for Early Platelet Activation
Associated with Mechanical Stimuli
[0037] Molecular markers, also referred to herein as molecular
features, for early platelet activation associated with mechanical
stimuli are described. The early activation markers can include an
elevated level of a negatively charge phospholipids, such as
phosphatidylserine and/or phosphatidylethanolamine on the external
surface of the platelets. Another early activation marker is an
elevated level of thrombin generation on the external surface of
the platelets. Optionally, a further early activation marker for
SMPA is no or a minimal change in P-selectin level, integrin
GPIIb/IIIa level, and/or integrin GPIIb/IIIa activation level
compared to a control from a healthy subject with minimal risk of
developing thrombosis.
[0038] In some embodiments, molecular signatures of mechanically
activated platelets include one or more molecular features of (a)
an elevated phosphatidylserine level on the external surface of the
platelets relative to a level of a control; (b) an elevated
thrombin level on the external surface of the platelets relative to
the level of the control; (c) an integrin GPIIb/IIIa level on the
external surface of the platelets and integrin GPIIb/IIIa
activation level, wherein these levels are not increased compared
to the level of the control; (d) a glycoprotein GP Ib level on the
external surface of the platelets, wherein the level is not
increased compared to the level of the control; (e) a P-selectin
level on the external surface of the platelets, wherein the level
is not increased compared to the level of the control; (f) a
decrease in platelet size relative to the platelet size of the
control; (g) an increase in microparticle generation relative to
the microparticle generation of the control, and (h) a change in
membrane lipidomic profile compared to the membrane lipidomic
profile of the control.
[0039] The disclosed methods can include measuring one or more
molecular markers in test platelets and comparing the level of the
marker(s) to a control. As illustrated in the experiments below,
the disclosed molecular signatures may vary somewhat from
cell-to-cell within a population of platelets. Thus, the disclosed
molecular signatures are typically used to characterize a
population of platelets, rather than an individual isolated
platelet. Methods that include measuring one or more molecular
markers may include measuring the one or more molecular markers on
a test population of platelets and determining the percentage of
platelets in the test population that are positive or negative for
the one or more molecular markers, and comparing the results to a
control population.
[0040] Individual platelets can be positive or negative for a
particular molecular marker when the platelet's level of expression
meets or exceeds a threshold level, or fails to meet or exceed the
threshold level, respectively. For example, in the experiments
below, the molecular marker on individual platelets is typically
measured by flow cytometry. Individual platelets are identified as
positive for a molecular marker when the fluorescence intensity
indicative of the molecular marker meets or exceeds a threshold
level of fluorescence.
[0041] Flow cytometry and other means of measuring a marker in
non-lysed cells are preferred methods of measuring expression of
the disclosed markers, particularly when localization (e.g., to the
cell surface) is important for the analysis.
[0042] If the percentage of the test population that is positive
for the molecular marker is higher than the percentage of the
control that is positive for the molecular marker, the marker can
be said to be elevated, higher, or increased relative to the
control population. If the percentage of the test population that
is positive for the molecular marker is lower than the percentage
of the control that is positive for the molecular marker, the
marker can be said to be lower or decreased relative to the control
population.
[0043] Molecular markers can also be measured by quantitating the
level of the marker(s) in a population. For example, in some
embodiments, the level of a molecular marker in a population is
determined by measuring its gene or protein expression.
[0044] If level of the molecular marker in the test population is
higher than the level of the marker in a quantitatively same or
similar control population, the marker can be said to be elevated,
higher, or increased relative to the control population. If level
of the molecular marker in the test population is lower than the
level of the marker in a quantitatively same or similar control
population, the marker can be said to be lower or reduced relative
to the control population.
[0045] The control can be a population of resting platelets
obtained from a healthy subject with minimal risk of developing
thrombosis. In some embodiments, a control is resting platelets
isolated from the subject under study. Phenotypes of resting
platelets are known in the art, for example, low levels of
P-selectin, integrin GPIIb/IIIa activation, phosphatidylserine,
thrombin generation, and/or a combination on the external
surface.
[0046] Additionally or alternatively, the test population can be
compared to a population of platelets treated with a chemical
activator. The chemical activator can have an effect on the
molecular marker relative to a control as described in the
disclosure and figures herein. Thus, the molecular signature of a
population of stress activated platelets can be distinguished from
that of both resting platelets and chemically activated platelets,
particularly when the signature is formed of two, three, four, or
more markers.
[0047] 1. Negatively Charged Phospholipids
[0048] Platelet membrane reorganization and externalization of
negatively charged phospholipids is considered as one of the latest
events of platelet activation, known as platelet procoagulant
activity. These negatively charged phospholipids, typically
including phosphatidylserine and phosphatidylethanolamine, cluster
and provide a surface for the assembly of tenase and prothrombinase
complexes, and hence catalyze local thrombin generation on the
platelet membrane.
[0049] Levels of one or more negatively charged phospholipids can
be elevated in mechanically activated platelets. For example, the
levels of one or more negatively charged phospholipids in the
mechanically-activated platelets can be elevated relative to a
control, such as a population of resting platelets obtained from a
healthy subject with minimal risk of developing thrombosis.
[0050] In some embodiments, one or more markers of molecular
signatures of mechanically activated platelets include one or more
negatively charged phospholipids, for example, phosphatidylserine.
In some embodiments, the level of phosphatidylserine in the
mechanically-activated platelets is elevated relative to a control.
In further embodiments, the level of phosphatidylserine on the
external surface of the mechanically activated platelets is
elevated than platelets activated by chemical activators such as
physiologically relevant soluble agonists e.g., Adenosine
DiPhosphate (ADP), epinephrine, thrombin receptor activating
peptide-6 (TRAP-6), thrombin, and collagen.
[0051] In some embodiments annexin V binding is used to measure
phosphatidylserine level. This assay is based on high affinity
binding of protein annexin V to phosphatidylserine on platelet
surface.
[0052] In some embodiments, two subpopulations can be distinguished
using an annexin V binding assay: a high annexin V sub-population
P3 (fluorescence intensity >4000 AU) and a low annexin V
sub-population P4 (fluorescence intensity 500-4000 AU). Thus, in
some embodiments, an individual platelet is considered to be
positive or high for phosphatidylserine when its fluorescence
intensity in an annexin V binding assay is greater than 4000 AU,
and negative or low for phosphatidylserine when its fluorescence
intensity in an annexin V binding assay is less than or equal to
4000 AU.
[0053] In some embodiments, the threshold for low annexin V
platelet sub-population is established based on its fluorescence
intensity level which must be higher than platelet spontaneous
fluorescence (i.e. fluorescence of intact platelet sample to which
no fluorescent dye-conjugated annexin V was added).
[0054] 2. Thrombin Generation from Platelets
[0055] In some embodiments, the methods can include measuring
thrombin generation on platelet surfaces to monitor platelet
procoagulant activity.
[0056] Levels of thrombin generation on platelet surfaces can be
elevated in mechanically activated platelets. In some embodiments,
the level of thrombin in the mechanically-activated platelets is
elevated relative to a control, such as a population of resting
platelets obtained from a healthy subject with minimal risk of
developing thrombosis. In further embodiments, the level of
thrombin on the external surface of the mechanically activated
platelets is elevated than platelets activated by chemical
activators such as physiologically relevant soluble agonists e.g.,
Adenosine DiPhosphate (ADP), epinephrine, thrombin receptor
activating peptide-6 (TRAP-6), thrombin, and collagen.
[0057] In some embodiments, thrombin generation rate is measured
using a chromogenic prothrombinase-based Platelet Activation State
(PAS) assay. The control can be thrombin generation rate on a
population of resting platelets obtained from a healthy subject
with minimal risk of developing thrombosis. In some embodiments, a
control is a thrombin generation rate on resting platelets isolated
from the subject under study.
[0058] 3. Surface Membrane Glycoprotein
[0059] In some embodiments, one or more markers of molecular
signatures of mechanically activated platelets include one or more
integrin of platelets, for example, Glycoprotein Ib (GPIb), also
known as CD42.
[0060] In some embodiments, the expression level of GPIb on the
external surface of the mechanically activated platelets is reduced
compared to expression level of GPIb of resting platelets, for
example, 5%, 10%, 15%, and 20%.
[0061] In preferred embodiments, the of GPIb on the external
surface of the mechanically activated platelets is about the same
or less than those activated by one or more chemical activators
such as physiologically relevant soluble agonists e.g., Adenosine
DiPhosphate (ADP), epinephrine, thrombin receptor activating
peptide-6 (TRAP-6), thrombin, collagen, and arachidonic acid.
[0062] 4. Selectins
[0063] P-selectin functions as a cell adhesion molecule (CAM) on
the surfaces of activated platelets. P-selectin mediates rolling of
platelets on activated endothelial cells. After platelet
activation, P-selectin is translocated from intracellular granules
to the external membrane, whereas fibrinogen aggregates platelets
by bridging integrin GPIIb/IIIa between adjacent platelets.
[0064] The methods can include measuring the P-selectin level on
the surface of platelets activated by shear stress. Levels of
P-selectin in a population of platelets that have been exposed to
high shear stress can be similar to that of a control, such as a
population of resting platelets obtained from a healthy subject
with minimal risk of developing thrombosis.
[0065] In some embodiments, the expression level of P-selectin on
the external surface of the mechanically activated platelets is
reduced compared to expression level of P-selectin of resting
platelets, for example, 5%, 10%, 15%, and 20%. In some embodiments,
the percentage of mechanically activated platelets positive for
P-selectin is the same, similar, or only slightly increased
relative to the percentage of resting platelets that are P selectin
positive, for example, an increase in the P-selectin positive
population of 35%, 30%, 25%, 20%, 15%, 10%, 5% or less than 5%
relative to the resting platelets population.
[0066] In preferred embodiments, P-selectin on the external surface
of the mechanically activated platelets is about the same or less
than those activated by chemical activators such as physiologically
relevant soluble agonists e.g., Adenosine DiPhosphate (ADP),
epinephrine, thrombin receptor activating peptide-6 (TRAP-6),
thrombin, collagen, and arachidonic.
[0067] In some embodiments, P-selectin exposure is detected by flow
cytometry. Platelets having high fluorescence intensity can be
identified as P-selectin positive platelets. In some embodiments,
an individual platelet is identified as P-selectin high or positive
when the fluorescence of the individual platelet is greater than or
equal to 3000 AU. Similarly, an individual platelet can be
identified as negative or low for P-selectin when the fluorescence
of the individual platelet is less than 3000 AU.
[0068] In some embodiments, the threshold for low P-selectin
platelet can be established based on its fluorescence intensity
level which must be higher than platelet spontaneous fluorescence
(i.e. fluorescence of an intact platelet to which no fluorescent
dye-conjugated anti-P-selectin antibody was added).
[0069] 5. Integrins
[0070] Platelets contain five integrins, three .beta.1 integrins
that mediate platelet adhesion to the matrix proteins collagen,
fibronectin and laminin, and the .beta.3 integrins .alpha.v.beta.3
and .alpha.IIb.beta.3 (J Clin Invest, 2005; 115: 3363).
[0071] In some embodiments, one or more markers of molecular
signatures of mechanically activated platelets include one or more
integrins of platelets, for example, glycoprotein (GPIIb/IIIa, also
known as integrin .alpha.IIb.beta.3).
[0072] In some embodiments, the expression level and/or activation
of integrin GPIIb/IIIa on the external surface of the mechanically
activated platelets is reduced compared to expression level of
integrin GPIIb/IIIa of resting platelets, for example, 5%, 10%,
15%, and 20%.
[0073] In some embodiments, the percentage of mechanically
activated platelets positive for expression and/or activation of
integrin GPIIb/IIIa is the same, similar, or only slightly
increased relative to the percentage of resting platelets that are
integrin GPIIb/IIIa positive, for example, an increase in the
integrin GPIIb/IIIa positive population of 35%, 30%, 25%, 20%, 15%,
10%, 5% or less than 5% relative to the resting platelets
population.
[0074] In preferred embodiments, the level and/or activation of
integrin GPIIb/IIIa on the external surface of the mechanically
activated platelets is about the same or less than those activated
by one or more chemical activators such as physiologically relevant
soluble agonists e.g., Adenosine DiPhosphate (ADP), epinephrine,
thrombin receptor activating peptide-6 (TRAP-6), thrombin,
collagen, and arachidonic acid.
[0075] In some embodiments, GPIIb/IIIa activation is determined by
quantifying and comparing the number of all GPIIb/IIIa-positive
platelets with the number of platelets presenting the activated
form of the integrin on their surface. Within the parental
CD41-positive platelet population, CD41/CD61-positive cells can be
distinguished. In some embodiments, the populations are
distinguished based on their fluorescence intensity. Individuals
platelets having fluorescence intensity greater than or equal to
4000 AU are positive for activated GPIIb/IIIa. Individual platelets
having less than 4000 AU are negative for activated GPIIb/IIIa.
[0076] In some embodiments, the threshold for positive for
activated GPIIb/IIIa platelet can be established based on its
fluorescence intensity level which must be higher than platelet
spontaneous fluorescence (i.e. fluorescence of an intact platelet
to which no fluorescent dye conjugated anti-CD41/CD61 antibody was
added).
[0077] 6. Platelet Size and Shape
[0078] Further changes associated with mechanically activated
platelets can also include morphological changes, for example,
platelet size and shape. In some embodiments, one or more markers
of molecular signatures of mechanically activated platelets include
the size and/or shape of platelets. Standard assays for determining
size and/or shape of platelets are known in the art, see for
example in Example 2 and associated FIG. 6C. Exemplary assays
include flow cytometry (e.g., measuring forward scatter and side
scatter (FSC-A/SSC-A) characteristics) and microscopy.
[0079] In some embodiments, mechanically activated platelets
decrease in size compared to resting platelets, for example, by 5%,
10%, 15%, 20%, 30%, 40%, 50%. In some embodiments, the threshold
for control platelets size can be established based on FSC-A/SSC-A
characteristics of an intact platelet toward which no mechanical or
chemical stimuli were applied.
[0080] 7. Microparticle Generation
[0081] Activated platelets vesiculate to produce platelet
microparticles (PMPs), a heterogeneous population of small
membrane-coated vesicles, ranging from 0.1 to 1.0 .mu.m in diameter
(Shai et al., J. Proteom. 76 Spec No: 287-296 (20120; Hsu et al.,
Immunol. Lett. 150 97-104 (2013)). In some embodiments, one or more
markers of molecular signatures of mechanically activated platelets
include microparticle generation. Standard assays for determining
microparticle generation of platelets are known in the art.
Exemplary assays include flow cytometry (e.g., measuring forward
scatter and side scatter (FSC-A/SSC-A) characteristics),
microscopy, and density centrifugation.
[0082] In some embodiments, mechanically activated platelets
increase microparticle generation compared to resting platelets. In
some embodiments, mechanically activated platelets increase
microparticle generation compared to platelets activated by
chemical activators such as physiologically relevant soluble
agonists e.g., Adenosine DiPhosphate (ADP), epinephrine, thrombin
receptor activating peptide-6 (TRAP-6), thrombin, collagen, and
arachidonic acid. In some embodiments, the number of microparticles
is considered to be elevated if it exceeds the number of
microparticles in an intact platelet sample towards which no
mechanical or chemical stimuli were applied.
[0083] 8. Membrane Lipidomic Profile
[0084] Changes in the lipidomic profile of the membrane, i.e,
changes in specific lipid composition of the platelet membrane can
also occur in mechanically activated platelets. In some
embodiments, one or more markers of molecular signatures of
mechanically activated platelets include changes in lipidomic
profile of platelet membrane. Methods for measuring lipidomic
profile have been described in the art, such as shotgun lipidomics
using mass spectrometry. See for example, Sampaio J L et al., PNAS
Feb. 1, 2011. 108 (5) 1903-1907, and Hu Q et al., BBA Clin. 2016
December; 6: 76-81.
[0085] Some exemplary lipids for preparing a lipidomic profile of
platelets include phosphatidylserine, phosphatidylinositol,
phosphatidic acid, phosphatidylglycerol, phosphatidylcholine,
acyl-carnitines, and sphingomyelins. In some embodiments,
mechanically activated platelets have a changed composition (e.g.,
membrane composition) of one or more lipids of phosphatidylserine,
phosphatidylinositol, phosphatidic acid, phosphatidylglycerol,
phosphatidylcholine, acyl-carnitines, and sphingomyelins, relative
to, for example resting platelets and/or platelets activated by one
or more chemical activators. In some embodiments, C20 lipids,
arachidonate precursors, or a combination thereof are altered in
activated platelets.
III. Methods
[0086] Methods for creating a molecular signature of
mechanically-activated platelets and methods of using the molecular
signature of mechanically-activated platelets are described. The
molecular biomarkers of mechanically-activated platelets can be
used to guide diagnostic and therapeutic therapy, or in monitoring
the disease activities and responses to treatment.
[0087] A. Methods of Creating a Molecular Signature of
Mechanically-Activated Platelets
[0088] Methods of creating a molecular signature of
mechanically-activated platelets are provided. In some embodiments,
the methods of creating a molecular signature of platelets
activated by one or more mechanical stimuli include measuring a
composite of at least two of (a) phosphatidylserine, (b) thrombin,
(c) integrin GPIIb/IIIa activation, (d) glycoprotein GP Ib, (e)
P-selectin; (f) platelet size; (g) microparticle generation, and
(h) lipidomic profile of the membrane, preferably at least three of
(a)-(h), at least four of (a)-(h), at least five of (a)-(h), at
least six of (a)-(h), at least seven of (a)-(h), and all of
(a)-(h). In some embodiments, the platelets are isolated from a
blood sample of a subject.
[0089] Methods for identifying cause or mechanism of platelet
activation are also described. Particularly, methods of early
identification of platelets activated by one or more mechanical
stimuli are described. In some embodiments, platelets are
identified as activated by one or more mechanical stimuli, not
physiologically relevant soluble agonists e.g., Adenosine
diphosphate (ADP), epinephrine, TRAP-6, thrombin, collagen, and
arachidonic acid if the platelets have a molecular signature
including one or more molecular features of (a) an elevated
phosphatidylserine level on the external surface of the platelets
relative to a level of a control; (b) an elevated thrombin level on
the external surface of the platelets relative to the level of the
control; (c) an integrin GPIIb/IIIa level on the external surface
of the platelets, wherein the level is not increased compared to
the level of the control; (d) a glycoprotein GP Ib level on the
external surface of the platelets, wherein the level is not
increased compared to the level of the control; (e) a P-selectin
level on the external surface of the platelets, wherein the level
is not increased compared to the level of the control; (f) a
decrease in platelet size relative to the platelet size of the
control; (g) an increase in microparticle generation relative to
the microparticle generation of the control, and (h) a change in
membrane lipidomic profile compared to the membrane lipidomic
profile of the control. In preferred embodiments, platelets are
identified as activated by one or more mechanical stimuli, not
physiologically relevant soluble agonists if the platelets have a
molecular signature including at least three of (a)-(h), at least
four of (a)-(h), at least five of (a)-(h), at least six of (a)-(h),
at least seven of (a)-(h), and all of (a)-(h).
[0090] B. Methods of Diagnosing
[0091] In some embodiments, the disclosed methods are utilized to
diagnose a subject at risk of developing thrombotic events. In
preferred embodiments, the disclosed methods are utilized to
diagnose a subject at risk of developing thrombotic events from
mechanically activated platelets.
[0092] In some embodiments, methods for identifying a subject at a
risk of developing a thrombotic event include the steps of (a)
creating a molecular signature of the platelets in the blood sample
collected from a subject according to claim 1; and (b) identifying
the subject as positive for developing a thrombotic event if the
molecular signature of the platelets include one or more molecular
features of (a) an elevated phosphatidylserine level on the
external surface of the platelets relative to a level of a control;
(b) an elevated thrombin level on the external surface of the
platelets relative to the level of the control; (c) an integrin
GPIIb/IIIa activation level on the external surface of the
platelets, wherein the level is not increased compared to the level
of the control; (d) a glycoprotein GP Ib level on the external
surface of the platelets, wherein the level is not increased
compared to the level of the control; (e) a P-selectin level on the
external surface of the platelets, wherein the level is not
increased compared to the level of the control; (f) a decrease in
platelet size relative to the platelet size of the control; (g) an
increase in microparticle generation relative to the microparticle
generation of the control, and (h) a change in membrane lipidomic
profile compared to the membrane lipidomic profile of the control,
preferably two or more features of (a)-(h), three of (a)-(h), at
least four of (a)-(h), at least five of (a)-(h), at least six of
(a)-(h), at least seven of (a)-(h), and all of (a)-(h).
[0093] C. Providing a Prevention and Management Regimen to a
Subject
[0094] In some embodiments, the methods further include providing
an appropriate therapy or protocol for the subject, for example,
after reviewing the diagnostic results, to prevent or reduce the
severity of an oncoming thrombotic event. In further embodiments,
methods include treating a subject identified as positive for a
thrombogenic status. The methods can reduce or prevent the onset or
development of a thrombotic event, and/or treat, prevent or manage
one or more thrombotic events in the subject relative to an
untreated control subject.
[0095] The subjects of the disclosed methods can be an animal. The
subjects are most typically mammals, for example humans.
[0096] Methods of treatment, method of monitoring treatment of a
subject, and combinations are also provided. The methods of
treatment can include monitoring the treatment, and any of the
methods of monitoring can include treating the subject.
[0097] Typically, the methods of treatment include administered a
subject a mechanoceutical in an effective amount to alter the
molecular signature of platelets to one that reflects a less
mechanically activated state. For example, the amount can be
effective to reduce phosphatidylserine level on the external
surfaces and/or reduce thrombin level on the external surfaces the
subject's population of platelets; increase average platelet size
in the subject's population of platelets; reduce microparticle
generation in the subject's population of platelets; change the
membrane lipidomic profile in the subject's population of
platelets; or a combination thereof.
[0098] Methods for delivering an agent to a subject typically
include administering to the subject one or more mechanoceuticals
one or more times. The methods can also include creating a
molecular signature of the platelets in the blood sample collected
from the subject after administering the one or more
mechanoceuticals. In some embodiments, the methods include creating
a second, third, fourth, fifth molecular signature of the platelets
in the blood sample collected from the subject later time points.
The methods can include repeating the administration of the
mechanoceutical if the level of phosphatidylserine, the level of
thrombin, and/or the level of microparticle generation is reduced;
the platelet size is increased; and/or the membrane lipidomic
profile is changed; optionally the level of integrin GPIIb/IIIa
activation and/or level of P-selectin in the platelets of the
subject is not affected. In some embodiments, if the level of
phosphatidylserine, the level of thrombin, and/or the level of
microparticle generation is not reduced; the platelet size is not
increased; and/or the membrane lipidomic profile is not changed;
the dose, the frequency of administration of the mechanoceutical,
or a combination thereof is increased, or a different
mechanoceutial is administered to the subject. In some embodiments,
the subject has a blood-contacting medical device implanted in the
body prior to, contemporaneous with, simultaneous with, or after
the first treatment with the mechanoceutical.
[0099] Methods of monitoring the prophylactic treatment of a
subject are also provided. The methods can include, for example,
creating a molecular signature of the platelets in the blood sample
collected from a subject before treatment, treating the subject
with a mechanoceutical one or more times, and creating a molecular
signature of the platelets in the blood sample collected from the
subject after treatment. In some embodiments, treatment is repeated
if the level of phosphatidylserine, the level of thrombin, and/or
the level of microparticle generation is reduced; and/or the
platelet size is increased; and/or the membrane lipidomic profile
is changed; and/or optionally the level of integrin GPIIb/IIIa
activation, the level of integrin GP Ib, and/or level of P-selectin
in the platelets of the subject is not affected in the molecular
signature of the platelets in the blood sample collected after from
the subject after treatment relative to the molecular signature of
the platelets in the blood sample collected from the subject before
treatment. In some embodiments, if the level of phosphatidylserine,
the level of thrombin, and/or the level of microparticle generation
is not reduced; and/or the platelet size is not increased; and/or
the membrane lipidomic profile is not changed in the blood sample
collected from the subject after treatment relative to the
molecular signature of the platelets in the blood sample collected
from the subject before treatment the dose, the frequency of
administration of the mechanoceutical, or a combination thereof is
increased, or a different mechanoceutial is administered to the
subject. In some embodiments, the methods include creating a third,
fourth, fifth molecular signature of the platelets in the blood
sample collected from the subject later time points, alone or in
combination with one, two three, four, five, or more rounds of
treatment at the same or different doses or frequencies of the same
or a different mechanoceutical.
[0100] 1. Conditions to be Treated
[0101] The basic function of platelets is to rapidly bind to
damaged blood vessels, aggregate to form thrombi, and prevent
excessive bleeding. However, activated platelets also aggregate at
the site of atherosclerotic plaque ruptures and endothelial cell
erosion, stimulating thrombus formation and promoting
atherothrombotic disease. Thus, in some embodiments, the methods
are used to detect early platelet activation and/or pre-diagnose
thrombotic events in a subject at risk of developing one or more
thrombotic diseases.
[0102] In some embodiments, the subject to be treated is one with a
blood-contacting device, such as mechanical circulatory support
devices and ventricular assist devices (VADs).
[0103] Venous thromboembolism (VTE) is a common complication in
patients with malignant disease. Emerging data have enhanced the
understanding of cancer-associated thrombosis, a major cause of
morbidity and mortality in patients with cancer. In addition to
VTE, arterial occlusion with stroke and anginal symptoms is
relatively common among cancer patients, and is possibly related to
genetic predisposition. Several risk factors for developing venous
thrombosis usually coexist in cancer patients including surgery,
hospital admissions and immobilization, the presence of an
indwelling central catheter, chemotherapy, use of
erythropoiesis-stimulating agents (ESAs) and new molecular-targeted
therapies such as antiangiogenic agents. In some embodiments, the
methods are used in a subject with a proliferative disease, e.g.,
cancer, especially those at risk of developing cancer-associated
thrombosis
[0104] Inflammation shifts the hemostatic mechanisms in favor of
thrombosis. In some embodiments, the methods disclosed herein are
for use in a subject with inflammation, especially those with
increased thrombotic tendency. Exemplary systemic inflammatory
diseases characterized by thrombotic tendency, including but not
limited to Behcet disease (BD), antineutrophilic cytoplasmic
antibody-associated vasculitides, Takayasu arteritis, rheumatoid
arthritis, systemic lupus erythematosus, antiphosholipid syndrome,
familial Mediterranean fever, thromboangiitis obliterans (TAO) and
inflammatory bowel diseases.
[0105] 2. Mechanoceuticals
[0106] One or more mechanoceuticals can be administered to modify
properties of the surface of the platelets. For example, the
mechanoceuticals can be administered in an effective amount to
reduce membrane rigidity/stiffness and/or increase membrane
fluidity of platelets in the blood. Increasing membrane fluidity of
platelets is effective to "rubberize" the membrane, allowing it to
tolerate more exogenous physical force and/or shear, effectively
desensitizing it to these stimuli.
[0107] Compositions can be administered which include one or more
mechanoceuticals useful for the modulation of cellular processes
that contribute to onset and progression of thrombosis. In some
embodiments, the compositions contain an effective amount of the
mechanoceutical(s) to reduce the number of mechanically-activated
platelets in the blood. In some embodiments, the compositions
contain an effective amount of the mechanoceutical(s) to reduce
membrane rigidity/stiffness and/or increase membrane fluidity of
platelets in the blood.
[0108] Methods of measuring membrane stiffness have been described,
for example using Dielectrophoresis (DEP) and electro-deformation
(EDF), in which platelets are gently stretched by oscillating
(alternating) electrical fields, which deform the platelet and
provide a measure of its stiffness without inducing activation
(Leung S L et al., Ann Biomed Eng. 44(4):903-13 (2016)). In some
embodiments, the compositions are effective to reduce membrane
rigidity/stiffness of platelets to be lower than 5 kPa, lower than
3.5 kPa, or lower than 3.0 kPa.
[0109] i. Exemplary Mechanoceuticals
[0110] A variety of mechanoceuticals and methods for administering
the mechanoceuticals are described in WO 2015/113001 to Marvin
Slepian. Preferred agents are lipids and lipid related compounds.
Lipids may be broadly defined as hydrophobic or amphiphilic small
molecules; the amphiphilic nature of some lipids allows them to
form structures such as vesicles, multilamellar/unilamellar
liposomes, or membranes in an aqueous environment. Useful lipids
include fatty acids, glycerolipids, glycerophospholipids,
sphingolipids, saccharolipids, and polyketides (derived from
condensation of ketoacyl subunits); and sterol lipids and prenol
lipids (derived from condensation of isoprene subunits).
[0111] Agents that are capable of modulating membrane fluidity
include but are not limited to, dimethysulfoxide (DMSO),
gangliosides, interpolating lipids, hormones, or sterols,
cholesterol, cholesterol hemisuccinate, lidocaine, procaine, sex
steroids, phenytoin, Docohexanoic acid, cortisol, estradiol, PGE2,
progesterone, medroxyprogesterone, insulin, glucagon, atropine,
carbachol, lutropin, neuropeptide Y, thyroid hormone, aldosterone,
vasopressin, perylene, 9-(di-cyanovinyl) julodinine,
1,6-diphenyl-1,3,5-hexatiene (DPH), TMA-DPH, DPH-PA, cis and
trans-parinaric acid, polyunsaturated fatty acids, phospatidyl
choline, phospahtidylserine, phospatidyl inositol, insitol,
choline, cerebroside, glycoshpingolipids, and sphingomyelin, and
combinations thereof.
[0112] The mechanoceuticals can be administered alone, or in
combination with other agents such as a polymeric coating, agents
that alter submembrane assemblies, modulate intracellular
microfilament or microtubule function or agents that modulate
intracellular fluid content and tonicity. In a preferred
embodiment, the cholesterol administered is non-atherogenic. Agents
that can alter sub-membrane assemblies involved in
mechano-transduction include, but are not limited to NSAIDs, e.g.
sulindac sulfide, and phenolic antioxidants, caffeic acid phenethyl
ester (CAPE), which may modulate Focal Adhesion Kinase (FAK, also
known as PTK2 protein tyrosine kinase 2 (PTK2)) signaling. Weyant,
et al., "Colon cancer chemoprotective drugs modulate
integrin-mediated signaling pathways", Clin Cancer Res, 6:949
(2000). Similarly, resveratrol may modulate FAK as well.
[0113] Rho kinase inhibitors, e.g. Y-27632 including FAK, talin and
other linkage and phosphorylated proteins, may be included in the
compositions in an effective amount to limit shear-mediated
platelet activation, and administered to the cells.
[0114] Additional useful agents include, but are not limited to,
phenolic antioxidants, caffeic acid phenethyl ester (CAPE) FAK
signaling and modulating agents, resveratrol, Rho kinase
inhibitors, e.g. Y-27632, inhibitors or modulators of talin,
paxillin, and vinculin and similar submembrane mechnotrasnductive
proteins.
[0115] Agents that can modulate intracellular microfilament
function can be administered in an effective amount to increase the
microfilament assembly and/or integrity. Suitable agents that are
capable of modulating intracellular microfilament function include,
but are not limited to, cytochalasins (e.g. cytochalasin B),
concanavalin, vincristine, vinblastine, oryzalin, trifluralin,
taxol, taxetere and similar compounds.
[0116] Agents that can modulate intracellular microtubule function
can be administered in an effective amount to increase the
microtubule assembly and/or integrity. Suitable agents that are
capable of modulating intracellular microtubule function include,
but are not limited to colchicine, colcemid, vinblastine,
vincristine, taxol, taxetere, 9-bromonscapine (EM011), docetaxel,
noscapinoids, and tau.
[0117] Optionally, the mechanoceutical compositions include one or
more agents in an effective amount to modulate intracellular fluid
content and tonicity. For example, the compositions may include
hypo or hypertonic solutions, for example, saline, lactated
ringers, dextrose, sucrose, mannitol, or similar small
molecules.
[0118] In these embodiments, the cells may be contacted (in vivo)
with or incubated ex vivo in hypo or hypertonic solution. The cells
may also be contacted with aquaporin receptors modulating agents,
agonists or antagonists, Hg C12, G-protein modulating agents,
vasopressin receptors modulators, including V1a, V1b and V2,
tolvaptan, conivaptan, and/or other vaptans.
[0119] ii. Methods of Administering the Mechanoceuticals
[0120] The mechanoceuticals can be included in a composition, along
with a suitable carrier.
[0121] The compositions may be administered by a variety of
suitable methods, including, but not limited to, orally,
systemically, locally, regionally, enterally, parenterally, and
subcutaneously. For example, a catheter can be inserted into a
patient, and the composition can be infused, perfused, or
superfused to the desired site to expose the cells thereto. For
regional delivery, the composition may be administered via an
osmotic pump.
[0122] D. Methods of Selecting a Medical Device
[0123] Methods for selecting a blood-contact medical device for
implanting into a subject in need thereof are provided.
[0124] Exemplary blood-contact medical devices include mechanical
circulatory support device, ventricular assist devices (VADs).
Various long-term implantable ventricular assist devices are
commercially available including those using Axial Flow (e.g.,
HEARTMATE II.RTM. by Thoratec, HEART ASSIST 5.RTM. by Reliant
Heart, JARVIK 2000.RTM. by Jarvik Inc), using Centrifugal Flow
(e.g., HVAD.RTM. by HeartWare, DURAHEART.RTM. by Terumo, HEARTMATE
III.RTM. by Thoratec), using mixed flow (e.g., SYNERGY.RTM. by
CircuLite, and MVAD.RTM. by HeartWare), and using flower maker
(e.g., INCOR.RTM. by Berlin Heart). Methods for selecting a
mechanical circulatory support device with optimal benefits and
minimal risks of device-related adverse events such as thrombosis
in a subject are described.
[0125] Typically, ex vivo methods for selecting a blood-contacting
medical device for implanting into a subject in need thereof
include the steps of (i) modeling a shear stress profile of a
blood-contacting medical device; (ii) reproducing the shear stress
profile of blood flow in a microfluidic device; (iii) flowing a
sample containing platelets collected from a subject through the
microfluidic device; (iv) creating a molecular signature of the
platelets by measuring a composite of at least two of (a)
phosphatidylserine, (b) thrombin, (c) integrin GPIIb/IIIa, (d)
glycoprotein GP Ib, (e) P-selectin; (f) platelet size; (g)
microparticle generation, and (h) lipidomic profile of the
membrane, preferably at least three of (a)-(h), at least four of
(a)-(h), at least five of (a)-(h), at least six of (a)-(h), at
least seven of (a)-(h), and all of (a)-(h). Typically, the device
for modeling a mechanical stimulus emulates thrombogenicity,
produces a probability density function of the blood-contacting
medical device, and determines trajectories for individual
particles flowing through the blood-contacting medical device.
[0126] The methods further involves the step of selecting a
blood-contacting medical device if one or more of the level of
phosphatidylserine, the level of thrombin, and the level of
microparticle generation is lower than the levels in the molecular
signature of mechanically activated platelets, if the platelet size
is greater than the platelet size in the molecular signature of
mechanically activated platelets; or a combination thereof;
optionally the level of integrin GPIIb/IIIa, the level of integrin
GP Ib, and/or the level of P-selectin in the platelets of the
subject is not different than each of these in the mechanically
activated platelets.
[0127] In some embodiments, the methods involve creating a
molecular signature of two, three, four, five or more
blood-contacting medical devices. The methods further include the
step of selecting a device with the lowest level of
phosphatidylserine on the external surface of the platelets, the
lowest level of thrombin on the external surface of the platelets,
and/or the lowest level of microparticle generation for implant
into the subject in need thereof.
[0128] E. Methods of Screening Mechanoceuticals
[0129] Routine clinical anti-platelet agents have limited efficacy
in modulating hypershear-mediated platelet activation associated
with mechanical circulatory support (Valerio L et al., Thromb Res.
163:162-171 (2018)). Thus, methods for screening mechanoceuticals
suitable for modulating hypershear-mediated platelet activation
associated with mechanical circulatory support are provided.
[0130] Generally, screening mechanoceuticals under a mechanical
stimulus similar to that in a subject at risk of developing a
thrombotic even is desirable, for example in a microfluid device
modeled with the shear stress profile of a subject at risk of
developing a thrombotic event. Similar microfluidic devices have
been described previously, for example in U.S. Publication No.
2017/0246632, and by Dimasi A et al. (Dimasi A et al., Med Eng
Phys. 48:31-38 (2017)).
[0131] Methods of selecting a mechanoceutical agent are also
provided. The methods can include, for example, reproducing a
mechanical stimulus profile of blood flow in a microfluidic device;
flowing a sample collected from the subject through the
microfluidic device in the presence and absence of a test agent,
wherein the sample comprises platelets separated from a blood
sample obtained from a subject; creating a molecular signature of
the platelets in the presence and absence of the test agent; and
selecting the test agent as a mechanoceutical if the level of
phosphatidylserine on the external surface of platelets, the level
of thrombin on the external surface of platelets, and/or the level
of microparticle generation is lower in the presence of the test
agent than in the absence of the test agent.
[0132] F. Controls
[0133] The effect of mechanically activated platelets can be
compared to a control. For example, in some embodiments, the
control cells are platelets directly healthy subjects with minimal
risk of developing thrombosis.
[0134] One of skill can identify a suitable control or standard for
use the methods disclosed herein. For example, in some embodiments,
the control is a molecular signature of a population of resting
platelets, preferably prepared in the same or similar manner as the
signature of the test platelets subject to the disclosed methods.
In some embodiments, the control molecular signature is prepared in
parallel with the test signature. In other embodiments, the control
molecular signature is prepared at a different time from the test
signature. Thus, in some embodiments, the control is a reference
signature or indices otherwise associated with non-mechanically
active platelets. The platelets utilized for the control signature
can be, but need not be, derived or collected from the blood of the
same subject as the platelets utilized for the test signature. For
example, in some embodiments, the control platelets are collected
from any individual who has not had a medical device (e.g., a
blood-contacting medical device) implanted therein. In some
embodiments, the control platelets are collected an individual
before undergoing an implantation procedure (e.g., of an
implantation of a blood-contacting medical device), and test
platelets are collected from the same individual after undergoing
the implantation procedure.
[0135] The effect of a mechaniceutical agent can be compared to a
control. For example, in some embodiments, one or more of the
pharmacological or physiological markers or pathways affected by
mechaniceutical agent treatment is compared to the same
pharmacological or physiological marker or pathway in untreated
control cells or untreated control subjects. For example, cell
surface markers of platelets including phosphatidylserine,
thrombin, GP Ib, integrin GPIIb/IIIa activation, P-selectin are
measured in mechaniceutical agent treated cells or subjects and
compared to untreated cells or subjects. In some embodiments, an
untreated control is derived from the same source as the treated
sample, for example, cells isolated from a patient at a risk of
developing a thrombotic event. In some embodiments, the untreated
cells are platelets with a cardiovascular pathology having the
molecular signature of mechanically activated platelets.
[0136] The present invention will be further understood by
reference to the following non-limiting examples.
EXAMPLES
Example 1: Platelet P-Selectin Exposure Occurs after a Stimulation
with Biochemical Activators but not Uniform Continuous Shear
Stress
[0137] Methods
[0138] Blood Collection and Platelet Fraction Isolation.
[0139] All healthy volunteers gave their written informed consent
and claimed not to have taken antiplatelet medications two weeks
prior to blood draw. The study protocol was approved by the
Institutional Review Board at the University of Arizona concerning
human subjects. Blood was drawn from forearm by venipuncture via
21-gauge (0.8.times.19 mm) butterfly needle into 30 mL syringe and
transported in polypropylene conical tube containing 3 mL of acid
citrate dextrose anticoagulant solution (107 mM trisodium citrate,
60 mM citric acid, 199 mM glucose). Platelet-rich plasma (PRP) was
obtained by centrifugation of anticoagulated blood at 400 g for 15
minutes at room temperature with no brake applied. PRP, as a
supernatant, was collected and placed into a new tube with a
plastic transfer pipette. To get platelet-poor plasma (PPP) the
remaining blood was centrifuged at 1430 g for 20 minutes at room
temperature. Gel-filtered platelets (GFP) were isolated after PRP
filtration through Sepharose 2B column (35.times.250 mm)
equilibrated with HEPES buffer (10 mM HEPES, pH-7.4, 125 mM NaCl,
2.7 mM KCl, 2 mM MgCl, 0.5 mM NaH.sub.2PO.sub.4, 1 mM trisodium
citrate, 25 mM glucose and 0.1% BSA). Platelet count was quantified
with Z1 Coulter Particle Counter (Beckman Coulter Inc.,
Indianapolis, Ind.). Platelet fractions were stored and handled at
room temperature if not otherwise indicated.
[0140] Platelet Aggregation Induced with Biochemical Agonists
[0141] Platelet aggregation was assessed in PRP or GFP samples
employing dual channel optical aggregometer (Model 560-VS,
Chrono-Log Corporation, Havertown, Pa.). 10 uM ADP (Sigma-Aldrich,
St. Louis, Mo.), 10 .mu.g/mL epinephrine (Sigma-Aldrich, St. Louis,
Mo.), 32 .mu.M TRAP6 (Roche Diagnostics GmbH, Mannheim, Germany), 1
U/mL thrombin (Sigma-Aldrich, St. Louis, Mo.), 100 .mu.g/mL
collagen (MP Biomedicals, Solon, Ohio) or 1 mM arachidonic acid
(BioData, Horsham, Pa.) were utilized to induce platelet
aggregation response. Platelet count in aggregation mixture was
adjusted to 150-200.times.10.sup.3 plt/.mu.L (in GFP) and to
300-350.times.10.sup.3 plt/.mu.L (in PRP), final CaCl2
concentration was 1 mM. Aggregation was started by adding mentioned
agonists and running for 5 minutes at 37.degree. C. with 1000 rpm
stirring. The changes in relative light transmittance were
recorded, and arbitrary values for each aggregation curve were
calculated with Aggrolink software (Chrono-Log Corporation,
Havertown, Pa.).
[0142] Shear-Mediated Platelet Activation
[0143] Platelets were exposed to uniform continuous shear stress in
the hemodynamic shearing device (HSD), a computer-controlled
cone-plate-Couette viscometer, specifically designed to generate
precisely controlled and uniformly distributed shear stress to all
platelets in the flow field (Nobili et al., ASAIO J. (American Soc.
Artif. Intern. Organs), vol. 54, no. 1, pp. 64-72, (2008)). GFP
were diluted with HEPES buffer to final platelet count
2.times.10.sup.4 platelets/.mu.L, and CaCl2 (2.5 mM) was added 3 mM
prior to HSD run. Constant shear stress modes of three magnitudes
(30, 50 and 70 dynes/cm.sup.2) were utilized. Samples for flow
cytometry and platelet activation state (PAS) assay were taken from
the annular region of the HSD at 0 and 10 minutes time points.
[0144] To emulate dynamic shear pattern found within MCS
conditions, an in vitro VAD-employed loop system was built using
axial continuous flow VAD HeartAssist 5 (MicroMed Technology Inc.,
USA) and 1/2'' diameter non-thrombogenic tubing. Outflow
graft-aorta anastomotic angle of 90 degrees was emulated by
insertion of angulated connector. GFP (2.times.10.sup.4
platelets/.mu.L, 2.5 mM CaCl.sub.2) were circulated through the VAD
loop system at 8000 rpm at room temperature, samples were collected
at 0, 2, 5, 10, 30, and 60 minutes time points, and processed
immediately.
[0145] To establish a positive control for SMPA and a reference
maximum for annexin V binding and PAS values, fully activated
platelets were obtained by sonication (10 W for 10 s, Branson
Sonifier 150 with microprobe, Branson, Mo., USA) (Schulz-Heik et
al., Pathophysiol. Haemost. Thromb., vol. 34, no. 6, pp. 255-262,
June (2006)). Sonication mixture (350 .mu.L) contained
2.times.10.sup.4 platelets/.mu.L and 2.5 mM CaCl.sub.2.
[0146] Flow Cytometry Detection of Platelet Activation State
Markers
[0147] To induce biochemical activation, GFP (2.times.10.sup.4
platelets/.mu.L, final volume 100 .mu.L) were treated with
biochemical agonists mentioned above in presence of 2.5 mM
CaCl.sub.2, mixed gently and incubated undisturbed for 15 minutes
at room temperature. Then APC-conjugated anti-CD62P (clone
Psel.KO2.3, eBioscience, San Diego, Calif.) and FITC-labeled
annexin V (eBioscience, San Diego, Calif.) were added to indicate
platelet .alpha.-granule secretion and phosphatidylserine
externalization (PSE) during platelet biochemical activation.
Integrin GP .alpha.IIb.beta.3 activation was evaluated by
double-staining technique. APC-conjugated anti-CD41 (clone MEM-06,
Thermo Scientific, Rockford, Ill.) bound to integrin subunit
.alpha.IIb presented on the surface of both intact and activated
platelets. FITC-anti-CD41/CD61 (clone PAC-1, BioLegend, San Diego,
Calif.) bound to activation-induced conformational epitope of the
integrin .alpha.IIb.beta.3 and mark only activated platelets. A
pair of antibodies against .alpha.IIb.beta.3 was added
simultaneously into a test tube containing activated platelet
sample to identify the number of platelets exposing activated
.alpha.IIb.beta.3 among all .alpha.IIb.beta.3-expressing platelet
population. Cells were incubated with annexin V or antibodies for
30 minutes at room temperature in the dark, washed by
centrifugation (5000 rpm, 5 min), resuspended in 1 ml of Ca-free
0.05 M phosphate buffer (pH--7.4) and transferred in polystyrene
tube for flow cytometry. Flow cytometry analysis was performed
within 15 minutes after sample preparation.
[0148] Shear-stimulated and sonicated platelet samples were
incubated with annexin V or fluorophore-labeled antibodies
immediately after HSD run or sonication procedure. To avoid
time-dependent fluorescence signal dissipation, those samples were
fixed with 3.5% PFA in PBS for 20 minutes at room temperature,
washed and resuspended in PBS as described above for biochemically
activated platelets.
[0149] Flow cytometry was performed on FACS Canto II (BD
Biosciences, San Jose, Calif.) using the following configurations:
HeNe red laser (633 nm) and 660/20 filter--for APC, solid state
blue laser (488 nm) and 530/30 filter--for FITC-stained cell
detection. Single platelets were distinguished from their
aggregates and microparticles based on their characteristic forward
versus side scatter. Percentage of activation was quantified as the
fraction of platelets that displayed greater than baseline levels
of annexin V binding and platelet activation state markers. BD
FACSDiva.TM. software was applied to analyze flow cytometry data
statistics (percentage of marker-positive platelets among whole
platelet population and their median fluorescence intensity).
[0150] Statistical Analysis
[0151] Flow cytometry and PAS assay samples were run in two
repetitions; results from four to six independent experiments with
different donors were summarized in plots. The data were
statistically analyzed using one-way analysis of variance (ANOVA)
from Microsoft Excel software package (Microsoft, Redmond, Wash.).
Averages are reported as the mean.+-.margin of error. The level of
statistical significance is indicated in the figures as p<0.05
(*) and p<0.01 (**).
[0152] Results
[0153] To define the molecular signature of SMPA, biochemical
versus mechanical platelet activation by uniform shear stress
applied in the hemodynamic shearing device (HSD) was compared. To
induce biochemical activation, human GFP were treated with the
panel of physiologically relevant soluble agonists--ADP,
epinephrine, TRAP-6, thrombin, collagen or arachidonic acid.
Agonists' effective concentrations were chosen considering their
ability to induce platelet aggregation in GFP (TRAP-6, collagen,
arachidonic acid, and thrombin) and/or PRP (ADP, epinephrine)
(FIGS. 1A-1F). Mechanical platelet activation was obtained via GFP
exposure to uniform continuous shear in HSD as described above in
"Shear-mediated platelet activation". Physiologically relevant
shear stress levels (30 and 50 dynes/cm.sup.2) close to those found
in blood stream vs. sub-pathological shear conditions (70
dynes/cm.sup.2) existing within MCS were examined Initially, the
P-selectin exposure, as a known marker of platelet activation and
.alpha.-granule secretion, was detected employing mono
immunostaining and quantified by flow cytometry. Those cells
showing high fluorescence intensity (.gtoreq.3000 AU) were gated as
P-selectin positive platelets and marked as sub-population P6
(FIGS. 2A-2G).
[0154] It was observed that all biochemical agonists induced
P-selectin exposure, but the extent of their action was notably
differed. In FIGS. 2A-2G, the significant shift of fluorescence
intensity peak to the right side after platelet biochemical
activation is present indicating the intensification of P-selectin
exposure on the surface of biochemically activated platelets.
"Weak" agonists, epinephrine (FIG. 2C) and ADP (FIG. 2B), induced
modest P-selectin exposure, the number of CD62P-positive cells was
18.6.+-.1.5% and 46.8.+-.3.9% of whole platelet population,
respectively. After arachidonic acid stimulation, 52.1.+-.6.8% of
cells presented P-selectin (FIG. 2G); TRAP-6 and thrombin promoted
the dramatic augmentation of P-selectin exposure (FIG. 2E), A clear
majority of platelets bared the protein marker on their surface.
Among "strong" agonists, only collagen failed to induce high
P-selectin exposure under experimental conditions employed (FIG.
2D).
[0155] The median fluorescence intensity of CD62P-positive
platelets grew at the same manner as their number in each
experimental group of cells treated with biochemical agonists (data
not shown).
[0156] Platelets subjected to all levels of uniform shear expressed
low levels of P selectin on their surface (FIGS. 3A-3F). As
compared with intact platelets, the number of CD62P-positive
platelets was not significantly increased after physiologically
relevant shear (FIGS. 3A-3B, groups "Intact platelets" vs. "30
dynes/cm.sup.2") and was approximately three times as high after
platelet exposure to relatively high shear stress, achieving
15.5.+-.3.6% and 18.8.+-.4.2% after 50 and 70 dynes/cm.sup.2,
respectively. Likewise, fluorescence intensity levels were barely
increased (data not shown). Sonicated platelets, established as a
positive control for mechanical platelet activation (Schulz-Heik et
al., Pathophysiol. Haemost. Thromb., vol. 34, no. 6, pp. 255-262,
June (2006)), also showed low level of P-selectin appeared on their
surface. Neither CD62P-positive platelet number nor their
fluorescence intensity were notably elevated as a result of
platelet sonication in presence of 2.5 mM CaCl.sub.2).
Example 2: Platelet Integrin GPIIb/IIIa Activation by Biochemical
Agonists Versus its Downregulation after Continuous Shear Stress
Results
[0157] To assess the magnitude of integrin GPIIb/IIIa activation
after platelet stimulation with constant shear or biochemical
agonists, the number of whole GPIIb/IIIa-positive platelet
population was compared with the number of those presenting
activated form of the integrin on their membrane. For this purpose,
double staining with APC-conjugated anti-CD41 and FITC-conjugated
anti-CD41/CD61 was applied as describe in the methods described
above. Among parental CD41-positive platelet population,
CD41/CD61-positive cells were distinguished based on their
FITC-fluorescence intensity level (.gtoreq.4000 AU) and assumed as
presenting activated form of integrin GPIIb/IIIa on their surface
(FIGS. 4A-4G, sub-population P3). The increase of fluorescence
intensity, as a significant right-side shift of fluorescence peak,
was notable only after ADP and thrombin stimulation of GFP. As
shown on FIGS. 4B and 4F, the number of platelets presenting
activated GPIIb/IIIa in ADP- and thrombin-treated cell groups was
elevated correspondingly in 4.2 and 9.6 times as compared with
intact platelets (28.0.+-.8.9% and 63.5.+-.10.2% vs. 6.7.+-.2.9%).
Surprisingly, other biochemical activators failed to promote
GPIIb/IIIa activation: the number of CD41/CD61-positive platelets
was not significantly elevated following GFP stimulation by
epinephrine (FIG. 4C), collagen (FIG. 4D), TRAP-6 (FIG. 4E), or
arachidonic acid (FIG. 4G).
[0158] Strikingly, platelet exposure to uniform continuous shear
stress did not promote integrin GPIIb/IIIa activation: the number
of platelets presenting activated form of GPIIb/IIIa was not
increased after 30, 50 or 70 dynes/cm.sup.2 shear stress levels
were applied. Similarly, in the sonicated platelet sample only
5.6.+-.3.3% of cells expressed activated GPIIb/IIIa on their
surface (FIGS. 5A-5F). Moreover, it was observed that platelet
exposure to high shear led to downregulation of GPIIb/IIIa surface
expression. As such, the number of GPIIb/IIIa-positive platelets in
GFP subjected to sub-pathological shear (70 dynes/cm.sup.2) was
minimally but statistically significantly decreased (88.0.+-.2.4%
vs. 93.8.+-.1.3% in "Intact platelets", FIG. 5D). Sonication
resulted in further deprivation of CD41-positive platelet count
down to 81.8.+-.3.3% (FIG. 5E). Following the same tendency, the
fluorescence intensity of CD41-positive platelets was also
decreased by 1.4 and 1.8 times after 70 dyn/cm.sup.2 shear stress
and sonication, respectively, as compared with intact platelets
(FIG. 5C).
[0159] It was found that after SMPA, the forward scatter (FCS-A) of
CD41-positive platelets, an indicator of their size, was distinctly
decreased as shear force magnitude increased (FIG. 6C), and the
extent of FCS-A reduction was comparable to fluorescence signal
dissipation.
Example 3: Phosphatidylserine Externalization and Thrombin
Generation on Platelet Surface as a Result of Shear-Mediated but
not Biochemical Activation
Methods
[0160] Platelet Activation State (PAS) Assay
[0161] PAS was quantified by chemically modified
prothrombinase-based chromogenic assay developed by Jesty and
Bluestein (Jesty & Bluestein, Anal. Biochem. (1999), Jesty et
al., Platelets, vol. 14, no. 3, pp. 143-149, 2003). The assay
measures the rate of acetylated thrombin generation from acetylated
proenzyme cleavage by factor Xa in presence of activated platelets.
Unlike native enzyme, acetylated thrombin possesses amidolytic
activity on peptide substrate but is not capable to activate
platelets in a feedback manner Activated, but not resting,
platelets present factor Va and negatively charged phospholipids
(mostly phosphatidylserine) on their surface for prothrombinase
complex formation and thrombin generation. Thus, the rate of
enzymatic cleavage of chromogenic substrate Chromozym TH
(Tosyl-Gly-Pro-Arg-4-nitranilide acetate, Roche Diagnostics GmbH,
Mannheim, Germany) by acetylated thrombin reflects the value of
initial activation state of the platelets tested (Jesty &
Bluestein, Anal. Biochem. (1999), Rubenstein et al., Circulation,
(2004)).
[0162] Platelet samples preliminary stimulated with biochemical
activators, shear or sonication (platelet count--5.times.10.sup.3
cells/.mu.L) were incubated with 200 nM acetylated prothrombin, 100
pM factor Xa (Enzyme Research Laboratories, South Bend, Ind.), 5 mM
CaCl.sub.2) in a volume of 100 .mu.L 20 mM HEPES, pH 7.4,
containing 130 mM NaCl and 0.1% BSA at 37.degree. C. for 10
minutes. Then, 10 uL of each timed sample was measured for thrombin
activity in microplate wells, containing 0.3 mM Cromozyme TH, 3 mM
EDTA in 20 mM HEPES, pH 7.4, containing 130 mM NaCl and 0.1% BSA.
Kinetic change of absorbance (.lamda.=405 nm) was measured for 7
minutes at room temperature using microplate reader Versa MAX
(Molecular Devices Corp., Sunnyvale, Calif.). The value of PAS or
initial rate of thrombin generation was calculated as a slope of
kinetic curve (.DELTA.A405/min) with SoftMax Pro6 Software.
[0163] Results
To detect phosphatidylserine exposure, an annexin V binding assay
is extensively utilized. The principle of the assay is based on the
high affinity binding of protein annexin V labeled with
fluorescence dye to phosphatidylserine on platelet surface with the
aid of Ca2+ ions (Demchenko, Cytotechnology, vol. 65, no. 2, pp.
157-72, March (2013)). In this study, annexin V binding was
measured to define the capability of biochemical agonists vs. shear
stress to induce platelet PSE and hence procoagulant activity. No
annexin V binding was observed after platelet stimulation with ADP,
epinephrine, collagen, TRAP-6, or thrombin taken alone (FIGS.
7A-7H), even though they induced aggregation, integrin GPIIb/IIIa
activation and/or P-selectin exposure. Arachidonic acid (1 mM) was
the only biochemical agonist shown to promote significant annexin V
binding: the number of annexin V-positive platelets reached
52.12.+-.6.79% of whole platelet population (FIG. 7G).
[0164] In contrast, platelets subjected to shear stress have shown
prominent annexin V binding capacity indicating the appearance of
phosphatidylserine on membrane surface. As shown in FIGS. 8A-8F,
the fluorescence intensity and the number of annexin V-positive
platelets were evidently increased after SMPA, the extent of
annexin V binding increase in parallel with the shear force
magnitude. Sonication, being a positive control for mechanical
activation, alike resulted in dramatic augmentation of annexin V
binding, approximately 50% of cells were found to expose
phosphatidylserine on their surface (FIG. 8E). It's noteworthy that
significant intensification of annexin V binding was observed even
after platelet activation by relatively low shear conditions, e.g.,
30 dynes/cm.sup.2 (FIG. 8B). The last observation denotes high
sensitivity of platelet membrane PSE to mechanical activation. The
distribution of fluorescence intensity within shear-treated
platelet groups appeared as high narrow peaks (FIGS. 8A-8E),
indicating a uniformity of PSE response to shear stress. The MFI
tended to increase with the shear magnitude, although the
significant change was shown only after platelet exposure to high
shear stress (FIG. 8G, "70 dynes/cm2" vs. "Intact platelets", ANOVA
p<0.01). Sonication, as positive control for mechanical
activation, indeed resulted in dramatic augmentation of annexin V
binding, with 45.02.+-.7.52% of cells exposing phosphatidylserine
on their surface (FIG. 8F).
[0165] The chromogenic PAS assay was applied to quantify the amount
of thrombin generated on activated platelet surface presenting
negatively charged phospholipids, and thus to measure the
procoagulant activity of platelets activated with biochemical
agonists or shear stress. Platelets stimulated with biochemical
mediators did not catalyzed prothrombin activation by factor Xa and
hence thrombin generation on platelet surface. Specifically, no
amidolytic activity was detected in presence of platelets
stimulated by ADP, epinephrine, collagen, or TRAP-6 (FIG. 9A). The
potency of arachidonic acid-treated platelets to force thrombin
formation was comparable with sonicated GFP sample, established as
positive control for SMPA.
[0166] Similar to the observation based on flow cytometry results
indicating PSE after SMPA, platelets subjected to shear stress have
expressed notable procoagulant activity (FIG. 9B). The rate of the
enzymatic reaction of prothrombin activation strongly correlated
with applied shear stress magnitude and reached 70.0.+-.1.2% of
sonication value when platelet stimulated by 70 dyn/cm.sup.2 shear
stress conditions (FIG. 9B). Platelets exposed to physiologically
relevant shear stress levels (30 and 50 dyn/cm.sup.2) indeed
expressed notable procoagulant activity.
Example 4: Molecular "Signature" of SMPA Under VAD-Generated
Dynamic Shear Stress within the Heart Assist V-Utilizing Model
Circulatory System
Results
[0167] To compare platelet function alterations underlying SMPA by
uniform constant shear stress applied in the HSD with actual
dynamic shear conditions of VAD-supported circulation, GFP was
recirculated through the VAD-utilizing loop system. Platelet
P-selectin exposure, as representative marker of platelet
biochemical activation, versus PSE and platelet prothrombinase
activity, found to depict early stages of mechanical activation,
were simultaneously evaluated. It was observed that platelets
experienced long-term dynamic shear stress in VAD loop system
showed exponential augmentation of annexin V binding over exposure
time (FIG. 10A). After 30 and 60 min passage, the number of annexin
V-positive platelets was significantly elevated and reached
3.3.+-.0.5% and 7.0.+-.0.7% as compared 0.5.+-.0.1% in non-sheared
sample, whereas no P-selectin exposure has been detected in those
platelet samples (FIG. 10C). Procoagulant activity of VAD
loop-sheared platelets was also elevated (FIG. 10B). As it has been
shown by PAS assay, the rate of prothrombin activation by factor Xa
was approximately 7-fold increased after 60 min shear exposure to
dynamic shear pattern of VAD-employed circulatory system as
compared with baseline.
[0168] This work has defined a molecular marker specific for SMPA
as compared with biochemical platelet activation by physiologically
relevant soluble agonists. The effect of physiological and
sub-pathological shear forces on human platelet activation was
investigated utilizing the hemodynamic shearing device (HSD), a
cone-plate-Couette viscometer, specifically designed to generate
precisely controlled and uniformly distributed shear stress to all
platelets in the flow field (Nobili et al., ASAIO J. (American Soc.
Artif. Intern. Organs), vol. 54, no. 1, pp. 64-72, (2008)). Applied
shear stress levels were chosen considering results of previous
numerical studies of VAD hemodynamics vs physiological levels of
shear existing within normal circulatory conditions (Nobili et al.,
ASAIO J. (American Soc. Artif. Intern. Organs), vol. 54, no. 1, pp.
64-72, (2008)). It has been shown that among examined platelet
activation markers (surface P-selectin exposure, integrin
.alpha.IIb.beta.3 expression and activation) none could reveal
ongoing SMPA. Alternatively, the extent of platelet annexin V
binding, reflecting anionic phospholipid externalization on
platelet surface and routinely applied to detect cell apoptosis,
positively correlated with level of shear and was detectable even
on initial stages of SMPA when low level of continuous uniform
shear was applied. After being identified for uniform continuous
shear stress conditions of the HSD, the SMPA signature has been
validated under highly dynamic shear environment in the
VAD-employing loop system, indicating its value as a diagnostic
signature for SMPA under MCS.
[0169] Therefore, this study defines specific markers of platelet
activation by shear stress as compared with biochemical activation
by physiologically relevant agonists. To induce SMPA in vitro,
platelets were exposed to either 1) uniform continuous shear in the
HSD (physiologically relevant and elevated shear stress levels were
tested), or 2) dynamic shear pattern of axial cfVAD-employed loop
system. The extent of platelet activation was monitored utilizing
both quantitative and functional approaches. Platelet activation
markers (P-selectin exposure and integrin .alpha.IIb.beta.3
activation) as well as annexin V binding reflecting PSE on platelet
surface were evaluated by flow cytometry. Additionally, platelet
procoagulant activity was assessed by chromogenic
prothrombinase-based PAS assay measuring thrombin generation on
platelet surface after their stimulation. It was found that
continuous shear stress (but not biochemical agonists) induced
prominent PSE and hence promoted thrombin generation on the
platelet surface. Both the extent of annexin V binding and the rate
of thrombin generation strongly correlated with the magnitude of
shear stress to which platelets were subjected. Simultaneously,
neither P-selectin exposure nor integrin .alpha.IIb.beta.3
activation usually accompanying biochemical platelet activation
were markedly elevated after SMPA. Moreover, even low levels of
continuous shear stress (30 and 50 dyn/cm.sup.2), when applied
uniformly to all platelets in the flow field of the HSD, resulted
in significant increase of both platelet PSE and procoagulant
activity. Further observation indicates that platelet membrane
lipid bilayer reorganization and procoagulant surface formation
occur (and could be detected) on early stages of SMPA when
platelets experience moderate shear stress, more likely existing in
vivo within highly dynamic flow conditions in VAD-supported
circulation. Thus, utilizing VAD-employed loop system, it was
verified that platelet long-term exposure to dynamic shear stress
indeed induced exponential elevation of PSE and thrombin generation
rates (as quantified by PAS assay), while P-selectin exposure
remained persistent over time. Both annexin V binding and PAS
levels were considerably lower as compared with those detected
within platelet activation by continuous shear (though
significantly increased versus non-sheared control). Observed
similarity of the molecular marker patterns for platelet activation
under dynamic and continuous shear stress conditions validates
feasibility of PSE as a specific and sensitive indicator of
platelet activation by shear stress.
[0170] Analyzing the competence of biochemical agonists to induce
platelet activation and PSE, it was shown that each of them
promotes distinctive pattern of molecular markers appearing on
platelet surface after the activation. Although all agonists were
potent drivers of platelet aggregation in GFP and/or PRP, their
ability to stimulate P-selectin exposure, integrin GPIIb/IIIa
activation and PSE varied drastically. This heterogeneity of
biochemical activation response reflects the diversity of signaling
pathways involved in its implementation. It was found that only ADP
and thrombin taken alone were capable to provoke high magnitude
P-selectin exposure and GPIIb/IIIa activation. Interestingly, both
ADP and thrombin simultaneously activate multiple types of
G-protein coupled receptors: ADP operates through purinergic
receptors P2Y1 and P2Y12 associated with Gq- and Gi-proteins, when
thrombin drives protease activated receptors PAR1 and PAR4
associated with Gq, Gi and G11. Such cooperative interaction
between Gq- and Gi-associated receptors' signals is needed to
enhance downstream platelet response and to promote long-term
activation events, i.e. integrin activation and aggregation which
require permanent platelet stimulation (Delaney et al.,
"Agonist-induced platelet procoagulant activity requires shear and
a Rac1-dependent signaling mechanism," vol. 124, no. 12, pp.
1957-1967, (2014), Ramstrom et al., Thromb. Haemost., vol. 89, no.
1, pp. 132-41, January (2003)). Therefore, TRAP6, specific PAR1
activator, as well as epinephrine, operating ultimately through
Gi-associated .alpha.2-adrenergic receptors, were failed to induce
full extent platelet activation. Stimulation by these agonists
resulted in P-selectin exposure, but no GPIIb/IIIa activation has
been detected. In the meantime, platelet incubation with
combination of sub-activating concentrations of ADP and epinephrine
led to notable integrin activation. It is worth highlighting that
neither thrombin nor ADP induced platelet PSE under the
experimental conditions, which indicates that additional
co-stimulatory support from other than G-protein signaling is
required to promote platelet membrane phospholipids' scrambling.
Taken as agonist of TXA2-operated pathway, arachidonic acid
appeared the only biochemical agent simultaneously provoking
notable PSE, P-selectin exposure and platelet aggregation. The
exclusive ability of arachidonic acid to induce such high extent
platelet response could be explained by its heterogenous effect on
cell functions. In human platelets arachidonic acid stimulates
enormous increase of intracellular calcium concentration, driven by
both Ca.sup.2+ influx through plasmatic membrane and its release
from intracellular stores (Alonso, Biochem. J, vol. 272, pp.
435-443, 1990)). While Ca.sup.2+ release could be prevented by
cyclooxygenase inhibitors and mimicked by TXA2 receptor agonist,
Ca.sup.2+ influx required higher AA concentration (60 uM) and was
not sensitive to those inhibitors. Recently, Rukoyatkina et al.
showed that sub-millimolar concentrations of arachidonic acid could
also induce the decline in platelet mitochondrial membrane
potential, increase in annexin V binding, and cleavage of
procaspase 3, similarly to known proapoptotic agent ABT-737
(Ramstro{umlaut over (m)} et al., Thromb. Haemost., vol. 89, no. 1,
pp. 132-41, January (2003)). In several cancer cell lines
TNF-alpha-induced elevation of intracellular arachidonic acid level
is also associated with the increase of mitochondria permeability,
release of cytochrome c, PSE, caspase 3 activation, following
chromatin fragmentation and rapid cell death.
[0171] In summary, these experimental findings showed that platelet
mechanical activation by shear stress, but not by vast majority of
biochemical agonists, induce PSE and thrombin generation on
platelet surface. Even dynamic shear conditions existing within
VAD-supported circulation result in notable increase of annexin V
binding. AA taken in sub-millimolar concentration was the only
agonist capable to provoke PSE. It is known that AA-evoked PSE
could be associated either with powerful Ca.sup.2+ signal driven by
this fatty acid or with platelet apoptotic response on high
concentration of AA. Which scenario of platelet response is
implemented under shear stress conditions remains unclear. The
incredible potency of low magnitude shear stress to induce fast PSE
indicates that signaling pathways other than biochemically evoked
are involved or an alternative amplification mechanism of platelet
response is employed.
[0172] Platelet membrane reorganization and following PSE occur as
a result of Ca.sup.2+ evoked scramblase activity disturbing
membrane lipid bilayer asymmetry and delivering aminophospholipids,
phosphatidylserine and phosphatidylethanolamine, from inner to
outer membrane leaflet. These negatively charged phospholipids
serve as high affinity binding sites for coagulation factors,
facilitating tenase and prothrombinase complexes assembly and,
thereby, catalyze thrombin generation on platelet surface. Platelet
PSE is not energy-consumable but indeed requires high and
persistent rise of intracellular Ca.sup.2+ concentration (up to 1
uM), hardly achieved under the platelet stimulation by
physiologically relevant biochemical agonists. As it was shown
lately (van der Meijden et al, Thromb. Haemost., vol. 93, no. 6,
pp. 1128-1136, (2005), Wolfs et al., C. Cell. Mol. Life Sci, vol.
6205, pp. 1514-1525, (2005), Delaney et al., "Agonist-induced
platelet procoagulant activity requires shear and a Rac1-dependent
signaling mechanism," vol. 124, no. 12, pp. 1957-1967, (2014)) and
confirmed in the study, platelet stimulation with agonists
operating either single (epinephrine, TRAP-6) or multiple (ADP,
thrombin) type of G-protein coupled receptors did not result in any
detectable annexin V binding, although successfully drove platelet
aggregation (under stirring conditions), integrin activation and/or
alpha-granule exocytosis. Thus, platelet agonist-stimulated PSE
requires simultaneous application of multiple biochemical agents or
shear force co-stimulation engaging alternative
Ca.sup.2+-mobilizing pathways.
[0173] Unless defined otherwise, all technical and scientific terms
used have the same meanings as commonly understood by one of skill
in the art to which the disclosed invention belongs. Publications
cited and the materials for which they are cited are specifically
incorporated by reference.
[0174] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
* * * * *